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] TP22JA15.043

    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] TP22JA15.044

    2.8 Spectrophotometric Analysis and Calculations
    2.8.1 Once a successful calibration curve for each of the two
reference oils has been created and verified, measure experimental
replicates for each of the reference oils at each temperature
followed by a standard check sample.
    2.8.2 The same procedure is followed for the positive controls.
    2.8.3 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.
    2.8.4 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] TP22JA15.045

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] TP22JA15.046

where:

[rho]Oil = density of the specific test oil, mg/mL and
VOil = Volume (mL of oil added to test flask (100 [mu]L =
0.1 mL))
    2.8.5 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.
    2.8.6 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] TP22JA15.047

where %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, t
n-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)    (%)
------------------------------------------------------------------------
ANS..................................................         5    >= 70
ANS..................................................        25    >= 75
IFO120...............................................         5    >= 55
IFO120...............................................        25    >= 65
------------------------------------------------------------------------

    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] TP22JA15.048

    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
                       Constituent                             (g/L)
------------------------------------------------------------------------
NaCl....................................................           21.03
Na2SO4..................................................            3.52
KCl.....................................................            0.61
KBr *...................................................           0.088
Na2B4O7 [middot]10H2O *.................................           0.034
MgCl2 [middot]6H20......................................            9.50
CaCl2 [middot]2H2O......................................            1.32
SrCl2 [middot]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
----------------------------------------------------------------------------------------------------------------
                                                               Viscosity @                        Category by
               Oil                Density, mg/mL  API gravity   15 [deg]C,   Category by API       kinematic
                                    @ 15 [deg]C      (deg)        (cSt)          gravity           viscosity
----------------------------------------------------------------------------------------------------------------
ANS.............................             884         28.2           40  Medium...........  Light
IFO120..........................             948         17.5         1520  Heavy............  Heavy
----------------------------------------------------------------------------------------------------------------

                   Table 3--Stock Solution Preparation
------------------------------------------------------------------------
                            Item                                Amount
------------------------------------------------------------------------
Mass of Bottle, g..........................................      29.9666
Mass of Bottle + oil, g....................................      31.5734
Mass of bottle + disp + oil + DCM, g.......................      55.0425
Mass of oil, g.............................................       1.6068
Mass of disp + oil + DCM, g................................      25.0759
Mass of 1 mL syringe, g....................................      14.5563
Mass of 1 mL syringe + solution, g.........................      15.8779
------------------------------------------------------------------------
Density of solution, g/mL..................................       1.3216
Volume of solution, mL.....................................      18.9740
Conc. Of stock solution, mg/mL.............................      84.6850
------------------------------------------------------------------------

                                                             [GRAPHIC] [TIFF OMITTED] TP22JA15.053

[GRAPHIC] [TIFF OMITTED] TP22JA15.049

    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 the purple sea urchin,
Strongylocentrotus purpuratus, (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        48-hr static      72-hr sea urchin   7-day subchronic:
          Test substance             acute: Menidia          acute:           Developmental    M. beryllina & A.
                                        beryllina      Americamysis Bahia         Assay              bahia
----------------------------------------------------------------------------------------------------------------
Dispersant only..................  yes...............  yes...............  yes...............  yes.
Dispersant/Reference Oil Mixture.  yes...............  yes...............  no................  no.
----------------------------------------------------------------------------------------------------------------

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 IFO 120 and ANS
oils. To obtain these oils at no charge (except for a minimal
shipping fee) see http://www.epa.gov/emergencies/content/ncp/index.htm. 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).
Repeat the following procedure for each reference oil tested. 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 seawater leaving a 20% headspace above
the liquid, place on a magnetic stir plate then add and center a
stir bar. Add the respective crude oil at 25g/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 1-2 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 [mu]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 1L 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).
    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 hr 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, (iii) egg production must occur in 50% of female Americamysis
bahia in the replicate control treatments.
    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
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                Subchronic M.                                 Development S.
                                       Acute M. beryllina        Acute A. bahia            beryllina         Subchronic A. bahia         purpuratus
--------------------------------------------------------------------------------------------------------------------------------------------------------
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      controls.
                                                                                where test starts      mg; >= 50% of
                                                                                with 7 day old         control females
                                                                                larvae, or >= 0.43     produce eggs.
                                                                                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 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
                                                   Solidfiers
----------------------------------------------------------------------------------------------------------------
                                                                   Test procedure
                                  ------------------------------------------------------------------------------
                                                                                48-hr static
     Application environment          96-hr static        48-hr static        96-hr static           acute:
                                     acute: Menidia          acute:         acute: Pimephales     Ceriodaphnia
                                        beryllina      Americamysis bahia       promelas             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 [mu]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
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                Freshwater Acute  P.
                                 Saltwater Acute  M. beryllina    Saltwater Acute  A. bahia             promelas             Freshwater Acute  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  2[permil]....  20  2[permil]...  NA..........................  NA.
                                ------------------------------------------------------------------------------------------------------------------------
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 [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......................  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
culture 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: Artificially distilled at
521[emsp14][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; 250 mL borosilicate glass Erlenmeyer
flasks;
    5.2.4 Pasteur pipettes;
    5.2.5 Multichannel pipettor (5-50 mL and 50-200 mL);
    5.2.6 Autoclave; environmental room or incubator;
    5.2.7 Balance accurate to 0.1 mg;
    5.2.8 Orbital shaker table with clamps sized to hold flasks
securely;
    5.2.9 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 a Agilent 6890 GC/5973 MS (or equivalent) equipped with an auto-
sampler for testing multiple samples; and
    5.2.10 Fixed Rotor Centrifuge.

5.3 Reagents and Culture Medium

    5.3.1 Stock Seawater Preparation. Prepare the artificial
seawater GP2 (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.

          Table 10--Artificial Seawater Nutrient Concentrations
------------------------------------------------------------------------
                                                               Final
                       Constituent                        concentration,
                                                                g/L
------------------------------------------------------------------------
* FeCl3 [middot] 6H2O...................................           0.050
KNO3....................................................           2.890
* Na5P3O10..............................................           0.297
------------------------------------------------------------------------
* Added aseptically after the GP2 has been autoclaved to limit
  phosphorus and iron precipitation.

    5.3.3 Seawater for bioremediation agents that do not include
nutrients. If a bioremediation agent contains living microorganisms
but not nutrients (or limiting concentrations of nutrients), then
nutrients may be added by the manufacturer. However, the total
concentration of the nutrients added to the bioremediation agent
must not exceed the final concentrations listed in Table 11 of this
Appendix.

Table 11--Artificial Seawater Nutrient Concentrations for Bioremediation
                   Agents Having No Nutrients Included
------------------------------------------------------------------------
                                                               Final
                       Constituent                        concentration,
                                                                g/L
------------------------------------------------------------------------
as Iron (Fe)............................................           0.010
as Nitrogen (N).........................................           0.400
as Phosphorus (P).......................................           0.075
------------------------------------------------------------------------

    If nutrients are supplied by the product manufacturer, the
specific composition and concentration used in the efficacy testing
must be submitted.
    5.3.4 Freshwater Preparation. The artificial freshwater, which
is a modification of Bushnell-Haas medium (Haines et al., 2005), is
prepared following the concentrations listed in Table 12 of this
Appendix and then autoclaved. The pH is adjusted to 7.4 before
autoclaving. Constituents removed from the original formulation are
KNO3, K2HPO4 and KH2PO4.

     Table 12--Constituent Concentrations for Artificial Freshwater
                             [Bushnell-Haas]
------------------------------------------------------------------------
                                                               Final
                       Constituent                         concentration
                                                              (mg/L)
------------------------------------------------------------------------
MgSO4 [middot] 7H2O.....................................             200
CaCl2 [middot] 2H2O.....................................              20
FeCl3 [middot] 6H2O.....................................              50
MnSO4 [middot] H2O......................................          0.0302
H3BO3...................................................          0.0572
ZnSO4 [middot] 7H2O.....................................          0.0428
(NH4)6Mo7O2.............................................          0.0347
------------------------------------------------------------------------

    5.3.5 Freshwater for the positive control. To prepare the
freshwater for the positive controls, prepare the nutrients
potassium phosphate monobasic (KH2PO4),
potassium phosphate dibasic (K2HPO4) and
potassium nitrate (KNO3) as a concentrated solution.
Filter sterilize and add to autoclaved artificial freshwater to
obtain the final concentrations given in Table 13 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
freshwater to 7.4 with 1 N HCl or 1 N NaOH, as appropriate.

              Table 13--Freshwater Nutrient Concentrations
------------------------------------------------------------------------
                                                               Final
                       Constituent                         concentration
                                                             (g/L) \1\
------------------------------------------------------------------------
KNO3....................................................            2.89
KH2PO4..................................................            1.00
K2HPO4..................................................            1.00
------------------------------------------------------------------------
\1\ Adjust pH to 7.4 prior to autoclaving

    5.3.6 Freshwater for bioremediation agents that contain living
microorganisms but not nutrients or limiting concentrations of
nutrients. If a bioremediation agent does not include nutrients,
then nutrients may be added. However, the total concentration of the
nutrients added to the bioremediation agent must not exceed the
final concentrations provided in Table 14 of this Appendix.

       Table 14--Artificial Freshwater Nutrient Concentrations for
           Bioremediation Agents Having No Nutrients Included
------------------------------------------------------------------------
              Constituent                  Final concentration, g/L \1\
------------------------------------------------------------------------
as Iron (Fe)...........................  not added since iron is already
                                          in the freshwater solution.
as Nitrogen (N)........................  0.400.
as Phosphorus (P)......................  0.400.
------------------------------------------------------------------------
\1\ Adjust to pH 7.4 prior to autoclaving.

    If nutrients are supplied by the product vendor, the specific
composition and concentration used in the efficacy testing must be
submitted.
    5.3.7 Oil Preparation. The test oil, weathered ANS521 crude oil,
can be obtained from EPA at no charge (except for a minimal shipping
fee). See http://www.epa.gov/emergencies/content/ncp/index.htm for
more information.
    5.3.8 Sodium azide sterilant. Prepare a stock solution of
NaN3 for addition to the negative killed control. The
final concentration in the killed controls will be 0.5 g/L.

5.4 Experimental Setup and Procedure

    5.4.1 Autoclave clean borosilicate glass Erlenmeyer flasks (250
mL) for 20 minutes at 121 [deg]C at 15 psig.
    5.4.2 Label flasks with the appropriate code (negative control,
positive control, or product; day to be sampled (0 or 28); letter
indicating replicate number) to reflect the following treatment
design in Table 15 of this Appendix:

  Table 15--Bioremediation Efficacy Test--Summary of Experimental Setup
------------------------------------------------------------------------
                                 Number of replicates at
                                     sampling times
           Treatment           --------------------------    Analysis
                                   Day 0        Day 28
------------------------------------------------------------------------
Negative (killed) Control (oil            0            3  GC/MS
 + exposure water + product +
 EPA culture + NaN3 sterilant).
*Positive control (oil +                  6            6  GC/MS
 exposure water + nutrients +
 EPA culture).
Test Type 1: Product                      6            6  GC/MS
 containing living
 microorganisms (oil +
 exposure water + living
 product + supplemented
 nutrients (if necessary)).
Test Type 2: Product                      6            6  GC/MS
 containing proprietary
 nutrients but no live
 microorganisms (oil +
 exposure water + product +
 EPA culture).
Test Type 3: Product (such as             6            6  GC/MS
 an enzyme) containing no live
 microorganisms and no
 nutrients (oil + exposure
 water + product).
------------------------------------------------------------------------
* The laboratory must report positive control test results conducted
  within the year of any test results for bioremediation products, for
  one or both types of water as applicable.

    5.4.3 Aseptically dispense 100 mL of pre-sterilized artificial
exposure water (seawater or freshwater) into each flask. For the
positive control flasks, use exposure water containing nutrients.
Alternatively, the artificial exposure water can be dispensed into
clean flasks and then autoclaved.
    5.4.4 Tare the labeled flasks containing exposure water and
other additions, as necessary, on the balance with a minimum
accuracy of 0.01 g. Add drop-wise 0.50 g oil (this results in a
final oil concentration of 5 g/L) using a sterile Pasteur pipette to
the center of the flask taking care to avoid splashing the oil onto
the sides of the flasks. Record the precise weight. ANS521 may be
previously warmed in a hot water bath to facilitate its flow. Take
precautions when handling and charging the flasks to minimize the
likelihood of contamination by exogenous microbes, including using a
new sterile pipette for each series of flasks.
    5.4.5 Preparation of the EPA culture for both the positive
control flasks and the flasks containing non-living bio-stimulation
products. Use two vials containing approximately 5 mL of the known
EPA culture frozen in glycerol. Thaw both vials at room temperature,
transfer the contents of both thawed vials to a single sterile
centrifuge tube, rinse tubes with two volumes each of sterile
exposure water, centrifuge at between 6,000 and 7,000 xs gravity
(6,000-7,000 x g) for 15 minutes using a fixed rotor to fully pellet
the cells.
    5.4.6 Positive control flasks contain exposure water, oil,
nutrients, and the EPA culture.
    5.4.7 Negative killed control flasks for all products shall
contain exposure water, oil, product, the EPA culture for products
not containing a living culture, and the sodium azide sterilant at a
final concentration of 0.5 g/L. Add the sodium azide sterilant prior
to adding any product or EPA culture. For the negative killed
control flasks and product flasks, prepare and add the product to
the flasks in a concentration specified by the manufacturer or
vendor.
    5.4.8 For non-living products that contain nutrient only, use
the EPA culture as the inoculum.
    5.4.9 For other non-living products (e.g., enzymes), do not add
nutrients or the EPA culture as the inoculum as they are not needed.
    5.4.10 For products containing microbial cultures, prepare 6
flasks the same way as in Steps a-d, but without the EPA culture. A
product that contains its own nutrients must not be amended with
nutrients, unless the product contains insufficient nutrients. Since
this is a closed flask test, nutrients could be limiting if they are
at the same concentration as used in the field. This could cause the
product to fail the test. Thus, the manufacturer has the option to
supplement its product with a higher concentration of nutrients than
that contained in the product. Any nutrient supplements to a product
must be reported and must not exceed the concentration limits in
Table 10 (for seawater) and 13 (for freshwater) of this Appendix, as
applicable.
    5.4.11 Cap all flasks either with cotton stoppers or loosely
applied aluminum foil to allow gas exchange with the atmosphere. Set
aside the T=0 flasks for immediate extraction and analysis. Place
the rest of the flasks onto the orbital shaker table. Do not tip the
flasks excessively to avoid stranding oil above the mixing area of
the flask. Set the orbital shaker to 200 rpm and shake the flasks
for 28 days at 20-23 [deg]C.
    5.4.12 Submit all information on added cultures and nutrients
for testing in the data report.

5.5 Sampling and Chemical Analysis

    5.5.1 Summary. At each sampling event (Days 0 and 28), product
and control flasks are sacrificed for analysis of residual oil
concentrations (SOP 4 of this Appendix). Record all physical
observations for each flask (such as degree of emulsification,
whether the oil has congealed into tar balls, wall growth, color,
etc.) at each sampling. The analytical procedure is summarized in
Table 16 of this Appendix. Dichloromethane (DCM) is the solvent used
for the initial extraction. Solvent-exchange the extract into hexane
prior to injection into the gas chromatograph. The solvent exchange
is done to prevent asphaltenes from contaminating the column.

                       Table 16--Bioremediation Efficacy--Summary of Analytical Procedures
----------------------------------------------------------------------------------------------------------------
                                                                        Sample
                                      Sampling/        Analysis       container/    Preservation/
    Matrix         Measurement       measurement        method       quantity of       storage     Holding times
                                       method                           sample
----------------------------------------------------------------------------------------------------------------
DCM...........  N/A.............  Solvent Exchange  N/A..........  Capped Vial      4 [deg]C.....  6 months.
                                   to Hexane.                       with Teflon
                                                                    septa, 30 mL.
Hexane........  Hydrocarbon       SOP 4...........  GC/MS........  Capped Vial      4 [deg]C.....  6 months.
                 Concentration.                                     with Teflon
                                                                    septa, 10 mL.
----------------------------------------------------------------------------------------------------------------

    5.5.2 Hydrocarbon Extraction. To measure extraction efficiency,
500 mL of the surrogate recovery standard (compounds and
concentrations described in SOP 1 in this Appendix) is added to each
flask. Add 50 mL DCM to each flask. Use magnetic stir bars to stir
the flasks for 20 minutes to transfer the oil into the DCM phase.
Allow the flasks to stand one hour for phase separation. If an
emulsion remains after one hour, centrifuge the emulsion in
Teflon[supreg] centrifuge tubes for at least ten minutes in a low
speed centrifuge at 3,000 x gravity (3,000 x g) to break the
emulsion and recover the DCM phase. Pass the DCM extract through a
funnel plugged with glass wool and containing approximately 20 g
anhydrous, granular sodium sulfate (Na2SO4) to
remove water. Collect the extract in 30 mL serum vials, capped with
Teflon lined septa and aluminum crimp seals, and store at 4 [deg]C
for up to 6 months.
    5.5.3 Solvent Exchange. Perform a solvent exchange (DCM to
hexane) prior to GC/MS analysis to prevent injection of asphaltenes
into the GC/MS column. Using a Class A volumetric pipette,
transfer10 mL of DCM extract to 16 x 125 mm test tubes with Teflon
lined screw caps. Place the test tubes in a 37 [deg]C water bath
under a stream of dry nitrogen gas. Reduce the sample to 1 mL and
return to volume with hexane. Repeat the blow down and re-suspension
twice to exchange the hydrocarbons from DCM into the hexane.
    5.5.4 Hydrocarbon Analysis. Quantify the concentrations of 25
alkanes (including hopane) and 31 aromatics (SOP 4, Table SOP 4.1 of
this Appendix) using an Agilent 6890 GC/5973 MS or equivalent
equipped with a 30-m x 0.25-mm ID x 0.25-[mu]m film thickness DB-5
or equivalent fused silica column. To prepare the samples, transfer
1.0 mL of the hexane extract into a 2 mL autosampler vial with
Teflon lined cap. Add 20 [mu]L of internal standard solution to each
vial with a syringe or positive displacement pipettor. SOP 2 of this
Appendix outlines the procedure for preparing the internal standard
solution. Load vials onto the autosampler tray and analyze in
selected ion monitoring mode (SIM). Sum the individual alkane
concentrations for the total alkane concentration and the individual
aromatic concentrations for total aromatic concentrations in each
flask.

5.6 Quality Assurance/Quality Control (QA/QC)

    5.6.1 Objectives. The critical variables to be analyzed for each
set of experimental conditions are the individual petroleum
hydrocarbons, i.e., the alkanes ranging in carbon number from nC-14
to nC-35, plus pristane and phytane, and the 2- to 4-ring polycyclic
aromatic hydrocarbons (PAHs) and their alkylated homologs as listed
in SOP 4 of this Appendix. The quality assurance objectives for
precision, accuracy, and detection limits are  20%, 75-
125% recovery, and 22.5 [mu]g/L, respectively. For more details,
refer to the SOPs of this Appendix.
    5.6.2 Precision Objectives. Precision is presented as relative
percent difference (RPD) for duplicate measurements and as relative
standard deviation (RSD, or coefficient of variance) for triplicate
measurements, applicable to replication of treatments as separate
samples.
    5.6.3 Accuracy Objectives. These are based on the check
standards and standard oil samples run concurrently with the sample
analyses for GC/MS analysis of critical

compounds. Critical compounds in the check standards and in the oil
standards must fall within 75-125% of expected values for the
analysis to be valid. Six surrogate compounds (SOP 1 of this
Appendix) added to each sample before extraction can also serve as a
surrogate for determining accuracy. The measured surrogate
concentrations must fall within 75-125% of expected values.
    5.6.4 Calibration Range. Conduct all measurements within the
linear calibration range of the instrument. The calibrated
concentration range for GC/MS analysis is 1 mg/L to 30 mg/L. If the
measured concentration of any critical compound is above the
calibration range, dilute the sample and re-analyze to quantify that
particular compound within the linear calibration range.
    5.6.5 Quality Control. Table 17 of this Appendix summarizes the
QC checks for each measurement. See the corresponding SOP in this
Appendix for detailed descriptions of QC checks, frequency,
acceptance criteria, and corrective actions.

                                             Table 17--QA/QC Checks
----------------------------------------------------------------------------------------------------------------
                                                                                Acceptance
  Sample matrix       Measurement        QA/QC check         Frequency           criteria      Corrective action
----------------------------------------------------------------------------------------------------------------
DCM.............  GC/MS hydrocarbon   Blanks...........  Once per           Peak area of       Flush with
                   analysis.                              calibrated run.    interfering        solvent, clean
                                                                             peaks <10% of      injection port,
                                                                             lowest standard    and/or bake
                                                                             peak area.         column.
DCM.............  GC/MS hydrocarbon   DFTPP Check        Once per           Must pass all      If any criteria
                   analysis.           Standard.          calibrated run.    DFTPP criteria.    fail, retune and
                                                                                rerun DFTPP
                                                                                check standard.
DCM.............  GC/MS hydrocarbon   Initial            Once per           Response Factor    If RSD for any
                   analysis.           Calibration        calibrated run.    RSD <=25%.         one compound
                                       Samples.                                                 >25%,
                                                                                recalibrate.
DCM.............  GC/MS hydrocarbon   Calibration Check  Every 10-15         25%   If >5 compounds
                   analysis.           Standards.         samples.           of expected        are out of
                                                                             values.            range,
                                                                                recalibrate and
                                                                                rerun samples.
Hexane..........  GC/MS hydrocarbon   Surrogates.......  Every Sample.....   30%   Re-inject.
                   analysis.                                                 of expected
                                                                             values.
Hexane..........  GC/MS hydrocarbon   Biomarker          Every Sample.....   25%   Re-inject.
                   analysis.           Concentration.                        of average
                                                                             values.
----------------------------------------------------------------------------------------------------------------

5.7 Pass/Fail Criteria

    5.7.1 Calculate the mean and standard deviation of the hopane-
normalized total aromatics (sum of all resolved aromatics) and
hopane-normalized total alkane concentrations (sum of all resolved
alkanes) from the 6 independent replicates at days 0 and 28. To
normalize, divide the sum of the alkane analytes and the sum of the
aromatic analytes in each replicate by the hopane concentration in
the corresponding replicate.
    5.7.2 From those data, calculate the 95% Upper Confidence Level
(UCL95) at days 0 and 28 using the following formula
(Equation 11 of this Appendix):
[GRAPHIC] [TIFF OMITTED] TP22JA15.050

Where:

xt28 = total hopane-normalized alkane or total hopane-
normalized aromatic mean of 6 replicates at days 0 and 28,
t95, 5 df = the 95% one-tailed t-value with 5 degrees of
freedom (2.015),
[sigma] = the standard deviation of the 6 replicates at day 0 and
28, and
n = no. of replicates = 6.

    5.7.3 Using Equation 12 of this Appendix, calculate the %
reduction of each oil fraction from day 0 to day 28, using the day 0
and 28 UCL95 hopane-normalized values for each fraction:
[GRAPHIC] [TIFF OMITTED] TP22JA15.051

Where:

t28(UCL95) = UCL95 of the hopane-normalized
total alkane or total aromatic mean of 6 replicates on day 28, and
t0(UCL95) = UCL95 of the hopane-normalized
total alkane or total aromatic mean of 6 replicates on day 0.

    5.7.4 A product is successful in saltwater if the % reduction of
total alkanes (aliphatic fraction) from the GC/MS analysis is
greater than or equal to 95% and the % reduction of total aromatics
(aromatic fraction) is greater than or equal to 70% at day 28 based
on the UCL95 (Equation 12 of this Appendix). For
freshwater, a product is successful if the % reduction of total
alkanes and total aromatics is greater than or equal to 95% and 40%,
respectively. The benchmark reduction ranges in aliphatic and
aromatic fractions for the positive control are the same as for the
products specified above. The average concentration of the biomarker
hopane at day 28 must not differ from the average concentration at
day 0 by more than 12% in the positive control. If the conditions
for the positive control are not met, the entire procedure must be
repeated.
    5.8 Data Verification and Reporting. GC/MS data files are
generated by MS ChemStation software (the Agilent standard software
for GC/MS) or equivalent for each injection. Data files contain
summed ion chromatograms and selected ion chromatograms. Calibration
curves are generated within MS ChemStation software, and all data
files are calculated against the calibration curve by MS
ChemStation. Data verification would be done by crosschecking
between analysts for 10% of the raw data and its reduction process.

    5.9 Laboratory Report. The summary of findings from a product
test must include the data listings for each analyte that was
analyzed (i.e., all individual alkanes and aromatics in the list of
required analytes). Express all concentrations as mg analyte/L
exposure water.

5.10 Standard Operating Procedures (SOPs) 1-4

    5.10.1 SOP 1. Preparation of Surrogate Recovery Standards
    <IP_ADDRESS> Preparation:
    <IP_ADDRESS>.1 Solvents: Dichloromethane (DCM), Optima (Fisher)
grade or equivalent.
    <IP_ADDRESS>.2 Reagents: D36-Heptadecane (C17), D50-Tetracosane
(C24), D66-Dotriacontane (C32), D10-1-Methylnaphthalene, D10-
Phenanthrene, D10-Pyrene, 5-beta-cholestane (coprostane).
    Note: Deuterated reagents are available from Cambridge Isotope
Laboratories, Andover, MA.
    <IP_ADDRESS>.3 Equipment: Micro-spatula, Small beakers, Glass
funnel, Analytical balance (0.0001g), Vials with Teflon-lined caps,
Teflon wash bottle with Optima grade DCM, Volumetric flask (250 mL),
class A, Pasteur pipettes.
    <IP_ADDRESS> Procedure:
    <IP_ADDRESS>.1 Using a calibrated analytical balance, weigh 100 mg
(0.100 g) of each reagent into separate 10-25 mL beakers.
    <IP_ADDRESS>.2 Dissolve the reagents in their beakers by adding 10
mL DCM. Use a Pasteur pipette to transfer the solutions to a single
250 mL volumetric flask.
    <IP_ADDRESS>.3 Wash the beakers 3 or 4 times with DCM. Use a Pasteur
pipette to transfer each of the washings to the 250 mL volumetric
flask.
    <IP_ADDRESS>.4 Dilute the solution to the 250 mL volume mark on the
volumetric flask with DCM.
    <IP_ADDRESS>.5 Use a glass stopper to seal the flask and homogenize
the solution by inverting the flask 5 or more times. The final
concentration of this solution is 400 mg/L for each of the reagents.
    <IP_ADDRESS>.6 Transfer the solution into 40 mL storage vials and
cap with Teflon-lined caps and label each with the date of
preparation, operator, sample names, and concentrations.
    <IP_ADDRESS>.7 Weigh each vial and record its weight on the label.
This weight is used to monitor possible evaporation during storage.
    <IP_ADDRESS>.8 Store these vials at 0 [deg]C or lower.
    <IP_ADDRESS>.9 Before using, allow the solution to come to room
temperature, and then shake it well.
    <IP_ADDRESS>.10 Weigh the vial before using it, and compare the
weight with the last weight recorded on the vial.
    <IP_ADDRESS>.11 If the weights are consistent, the integrity of the
solution can be assumed. If not, investigate and resolve the cause.
Prepare a new solution if the integrity has been compromised.
    <IP_ADDRESS> Quality Control: Inject 20 [mu]L of the surrogate stock
solution into 1 mL DCM. Add 20 [mu]L of the internal standard
solution (SOP 2 of this Appendix). Analyze this solution by GC/MS
using a calibrated method (SOPs 3 and 4 of this Appendix). The
expected concentration of each of the corresponding surrogate
compounds is 8  2 mg/L. If the measured value does not
fall within this range, prepare and measure another independent
surrogate solution. If the measured concentration of the second
surrogate solution is within the allowable tolerance range, the
calibration and instrument conditions are acceptable; properly
discard the first surrogate solution. If the concentration of the
second surrogate solution is also out of range, then clean and
recalibrate the instrument until the problem is resolved.
    5.10.2 SOP 2. Preparation of Internal Standard Solution

<IP_ADDRESS> Preparation

    <IP_ADDRESS>.1 Solvents: Dichloromethane (DCM), Optima (Fisher)
grade or equivalent.
    <IP_ADDRESS>.2 Reagents: D34 n-Hexadecane (C16), D42 n-Eicosane
(C20), D62 n-Triacontane (C30), D8-Naphthalene, D10-Anthracene, D12-
Chrysene, 5-alpha-Androstane.
    Note: Deuterated reagents are available from Cambridge Isotope
Laboratories, Andover, MA.
    <IP_ADDRESS>.3 Equipment: Micro-spatula, Small beakers, Glass
funnel, Analytical balance (0.0001g), calibrated and checked for
accuracy, Amber vials with Teflon-lined caps, labeled, Teflon wash
bottle with DCM, Volumetric flask (200 mL), class A, Pasteur
pipettes.
    <IP_ADDRESS> Procedure:
    <IP_ADDRESS>.1 Using a calibrated analytical balance, weigh 100 mg
(0.100 g) of each of the reagents into separate small beakers.
    <IP_ADDRESS>.2 Dissolve the reagents in their beakers by adding 10
mL DCM; using a Pasteur pipette, transfer the solutions to a single
200 mL volumetric flask.
    <IP_ADDRESS>.3 Wash the beakers 3 or 4 times with DCM; use a Pasteur
pipette to transfer each of the washings to the 200 mL volume mark
on the volumetric flask.
    <IP_ADDRESS>.4 Dilute the solution with DCM to the 200 mL volume.
    <IP_ADDRESS>.5 Seal the flask with a glass stopper and homogenize
the solution by inverting the flask a minimum of 5 times. The final
concentration of this solution is 500 mg/L of each reagent.
    <IP_ADDRESS>.6 Transfer the solution into 40 mL storage vials and
cap with Teflon-lined caps. Label each vial with the date of
preparation, operator, sample names, and concentrations.
    <IP_ADDRESS>.7 Weigh each vial, and record its weight on the label.
This weight is used to monitor possible evaporation during storage.
    <IP_ADDRESS>.8 Store this solution at 0 [deg]C or lower.
    <IP_ADDRESS>.9 Before using, allow the solution to come to room
temperature, and then shake it well.
    <IP_ADDRESS>.10 Weigh the vial before using it, and compare the
weight with the last weight recorded on the vial.
    <IP_ADDRESS>.11 If the weights are consistent, the integrity of the
solution can be assumed. If not, investigate and resolve the cause.
Prepare a new solution if the integrity has been compromised.
    <IP_ADDRESS> Quality Control: Inject 20 [mu]L of the internal
standard solution into 1 mL DCM. Analyze this solution by GC/MS. The
only peaks corresponding to the internal standards must appear. If
other peaks appear, particularly close to the internal standard
peaks, discard the internal standard solution and prepare a new
solution.
    5.10.3 SOP 3. Preparation of Working Standards, Check Standards,
and Oil Standards for GC/MS Consistency.
    <IP_ADDRESS> Preparation:
    <IP_ADDRESS>.1 Solvent: Dichloromethane (DCM), Optima (Fisher) grade
or equivalent.
    <IP_ADDRESS>.2 Stock solutions:
    <IP_ADDRESS>.2.1 Oil analysis standard: 44 compounds, 100 mg/L in
hexane/DCM (9:1), four, 1-mL vials required. Available from Absolute
Standards, Inc., Hamden, CT, Part # 90311.
    <IP_ADDRESS>.2.2 Nine compound PAH standard: 1,000 mg/L in DCM, one
vial. Available from Absolute Standards, Inc., Hamden, CT, Part #
90822.
    <IP_ADDRESS>.2.3 1,2-Benzodiphenylene sulfide, (synonym for
naphthobenzothiophene). Prepare a 2 mg/mL stock solution. Available
from Sigma-Aldrich Co., Part # 255122, purity 99%.
    <IP_ADDRESS>.2.4 Hopane solution (17 [beta] (H), 21[beta] (H), 0.1
mg/mL in isooctane. Available from Sigma-Aldrich Co. Part # 07562.
    <IP_ADDRESS>.2.5 Surrogate solution: 400 mg/L of each reagent in DCM
(see SOP 1 of this Appendix).
    <IP_ADDRESS>.2.6 Internal standard solution, 500 mg/L in DCM (see
SOP 2 of this Appendix).
    <IP_ADDRESS>.3 Alaska North Slope Crude Oil 521 (ANS521).
    <IP_ADDRESS>.4 Equipment:
    <IP_ADDRESS>.4.1 Glass storage vials with Teflon-lined caps (2 mL
and 40 mL capacity);
    <IP_ADDRESS>.4.2 Volumetric flasks, Class A, 5 mL, 10 mL, and 100
mL.
    <IP_ADDRESS>.4.3 Glass syringes capable of dispensing 25-500 mL with
an accuracy and precision of  1%, or equivalent.
    <IP_ADDRESS>.4.4 Wheaton repetitive dispenser, Model 411 STEP-PETTE
or equivalent.
    <IP_ADDRESS>.4.5 Teflon wash bottle filled with Optima grade DCM or
equivalent grade DCM.
    <IP_ADDRESS>.4.6 Pasteur pipettes.
    The volumes of stock solutions required to make the working
standards are listed in Table SOP 3.1 of this Appendix.

                                     Table SOP 3.1--Amount of Stock Solutions Required To Make the Working Standards
--------------------------------------------------------------------------------------------------------------------------------------------------------
              Stock standards                    A            B            C            D            E                                            F
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                Oil
                                              Analysis    Aromatics    1,2-Benzo-   Surrogate      Hopane
                                              Mix (44       Mix (9    diphenylene    solution     solution                                    ISTD (500
   Working standards concentration, mg/L     compounds,   compounds,    sulfide     (100 mg/L)   (100 mg/L)    Volumetric Flask Volume mL       mg/L)
                                             100 mg/L)    1000 mg/L)  (NBT) (2 mg/    [mu]L        [mu]L                                        [mu]L
                                               [mu]L        [mu]L      mL)  [mu]L
--------------------------------------------------------------------------------------------------------------------------------------------------------
STD 30 (no hopane)........................         1500          150           75          375            0  5.............................          100
STD 20 (5 mg/L hopane)....................         1000          100           50          250          250  5.............................          100
STD 10 (2.5 mg/L hopane)..................          500           50           25          125          125  5.............................          100
STD 5 * (1 mg/L hopane)...................          500           50           25          125          100  10............................          200
STD 5-Utility (1 mg/L hopane).............          500           50           25          125          100  10 (used for preparation of               0
                                                                                STD 2.5 & STD 1).
                                           -----------------------------------------------------------------                                ------------
STD 2.5 (0.5 mg/L hopane).................                           Use 5 mL of STD 5-Utility and dilute to 10 mL                                   200
STD 1 (0.2 mg/L hopane)...................                           Use 2 mL of STD 5-Utility and dilute to 10 mL                                   200
--------------------------------------------------------------------------------------------------------------------------------------------------------
* Make extra STD 5 for use as check standard.

    <IP_ADDRESS> Procedure for Working Standards and Check Standards:
    <IP_ADDRESS>.1 Label three 5 mL volumetric flasks as STD30, STD20,
STD10, and two 10 mL volumetric flasks as STD5, and STD5-utility.
    <IP_ADDRESS>.2 Add 1-2 mL of DCM to each volumetric flask.
    <IP_ADDRESS>.3 Using glass syringes, add the appropriate volume of
stock solution A (as listed in Table SOP 3.1 of this Appendix) to
the flasks labeled STD30, STD20, STD10, STD5, and STD5-utility.
    <IP_ADDRESS>.4 Wash the walls of the inner neck of the flasks with
several drops of DCM to rinse off the residue of the stock solution
into the flasks.
    <IP_ADDRESS>.5 Repeat Step 3 and Step 4 to dispense stock solutions
B-E (do not add stock solution F, internal standard solution, at
this step).
    <IP_ADDRESS>.6 Dilute to volume with DCM for all the above flasks,
seal with glass stoppers, and invert several times to homogenize the
solutions.
    <IP_ADDRESS>.7 Label two additional 10 mL volumetric flasks as
STD2.5 and STD1. Wet with 1-2 mL DCM.
    <IP_ADDRESS>.8 Dispense 5 mL of STD5-utility solution into flask
STD2.5, and 2 mL of STD5-utility solution into flask STD1.
    <IP_ADDRESS>.9 Dilute to volume with DCM, seal with glass stoppers,
and invert several times to homogenize the solutions.
    <IP_ADDRESS>.10 Using a 100 [mu]L glass syringe, dispense 100 [mu]L
of internal standard solution into flasks STD30, STD20, and STD10.
Dispense 200 [mu]L into flasks STD5, STD2.5, and STD1 to give a
final concentration of 10 mg/L internal standard.
    <IP_ADDRESS>.11 Seal with glass stoppers, and invert the flasks
several times to homogenize the solutions.
    <IP_ADDRESS>.12 Transfer the solutions into 2 mL storage vials, and
cap with Teflon-lined caps.
    <IP_ADDRESS>.13 Label each vial with date of preparation, analyst,
sample names, and concentrations.
    <IP_ADDRESS>.14 Weigh each storage vial and record its weight on the
label. This weight is used to monitor possible evaporation during
storage.
    <IP_ADDRESS>.15 Store this solution at 0 [deg]C or below.
    <IP_ADDRESS>.16 Before using, allow the solution to come to room
temperature, and shake it well.
    <IP_ADDRESS>.17 Weigh the vial before opening, and compare the
weight with the last weight recorded on the vial. If the weights are
consistent, the integrity of the solution can be assumed. If not,
investigate and resolve the cause. Do not use the solution if the
integrity has been compromised.
    <IP_ADDRESS> Procedure for Oil Standard. In a 100 mL volumetric
flask, weigh 0.500 g of the standard ANS521 crude oil, add 2 mL of
surrogate solution (see SOP 1 of this Appendix), and bring to volume
with DCM. Add 2 mL of internal standard solution (see SOP 2 of this
Appendix). Follow steps <IP_ADDRESS>.11 through <IP_ADDRESS>.17 of this SOP,
substituting 40 mL storage vials for the 2 mL vials.
    <IP_ADDRESS> Quality Control/Quality Assurance:
    <IP_ADDRESS>.1 Run the six standard solutions using the GC/MS method
(SOP 4) on a tuned GC/MS. Use the EnviroQuant software or equivalent
to calculate the average Relative Response Factor (RRF) and the
relative standard deviation (RSD) of the RRFs for each analyte over
the six concentrations. The RRF is defined as:
[GRAPHIC] [TIFF OMITTED] TP22JA15.052

    <IP_ADDRESS>.2 The RSD of the RRFs for all analytes must be 25% or
less according to EPA Method 525.2.
    5.10.4 SOP 4. GC/MS Method for the Analysis of Crude Oil
Samples.
    <IP_ADDRESS> Instrument Specifications:
    <IP_ADDRESS>.1 Use an Agilent 6890 GC coupled with an Agilent 5973
mass selective detector (MSD) and an Agilent 6890 series auto
sampler or equivalent, equipped with a DB-5 capillary column (30 m,
0.25 mm I.D., and 0.25 [mu]m film thickness) or equivalent, and a
split/splitless injection port operating in the splitless mode. Data
acquisition occurs either in the SIM (selected ion monitoring) mode
for quantitative analysis or in SCAN mode for qualitative analysis.
In SIM mode, the dwell time of each ion is set to be 10 milliseconds
and the ions are split up into groups by retention time. One way to
divide the ions is by retention time grouping as shown in Table SOP
4-1 of this Appendix. The number of ions in each ion group must be
constant, yielding the same scan rate for each group.

        Table SOP 4-1--Ions Associated With Retention Time Groups
------------------------------------------------------------------------
               Group                                Ions
------------------------------------------------------------------------
1.................................  57, 66, 128, 136, 142, 152, 156,
                                     166, 170, 184
2.................................  57, 66, 166, 170, 178, 180, 184,
                                     188, 192, 194, 198, 208
3.................................  57, 66, 178, 184, 188, 192, 194,
                                     198, 202, 206, 208, 212, 220, 226

4.................................  57, 66, 192, 198, 202, 206, 208,
                                     212, 216, 220, 226, 230, 234, 245
5.................................  57, 66, 191, 217, 228, 240, 242,
                                     248, 256, 262, 264, 270, 276, 284
------------------------------------------------------------------------

    <IP_ADDRESS>.2 Table SOP 4.2 of this Appendix summarizes the
instrumental conditions for crude oil analysis. Use only ultra-high
purity helium (99.999% pure) as the carrier gas. In series, connect
a moisture trap, an oxygen trap, and an organic trap to the carrier
gas line before it enters the column.

      Table SOP 4.2--Instrumental Conditions for Crude Oil Analysis
------------------------------------------------------------------------

------------------------------------------------------------------------
Instrument................................  Agilent 6890 Series II Gas
                                             Chromatograph (GC) with an
                                             Agilent 5973MSD and an
                                             Agilent 6890 auto sampler,
                                             or equivalent.
Column....................................  DB-5 capillary column (30 m,
                                             0.25 mm I.D., and 0.25-
                                             [mu]m film thickness) or
                                             equivalent.
Carrier Gas...............................  Helium, ultra-high purity
                                             grade (99.999%).
Inlet Temperature.........................  300 [deg]C.
Transfer Line (detector) Temperature......  310 [deg]C.
Oven Temperature Program..................  50 [deg]C for 4 minutes,
                                             then 7 [deg]C/min to 310
                                             [deg]C, hold for 18
                                             minutes.
Flow Rate.................................  Constant flow at 1mL/min.
                                             Linear velocity: 36.2 cm/
                                             sec.
Injection Volume..........................  1 [mu]L.
Split/Splitless Mode......................  Splitless.
Total Run Time............................  59.18 minutes.
------------------------------------------------------------------------

    <IP_ADDRESS> Procedure for preparing the instrument:
    <IP_ADDRESS>.1 Lower the injection port temperature and the oven
temperature to 50 [deg]C or less to avoid oxidation of the column.
    <IP_ADDRESS>.2 Replace the liner with a clean, silanized liner. Do
not touch the liner with bare fingers. A small piece of muffled
glass wool may be inserted to protect the column.
    <IP_ADDRESS>.3 Return the injection port and oven to the appropriate
temperatures.
    <IP_ADDRESS>.4 Wait five minutes after the temperature equilibrates
before using the instrument.
    <IP_ADDRESS> Procedure for tuning the MSD:
    <IP_ADDRESS>.1 Perform an air/water check. The value reported for
the relative abundance of water (m/z 18), nitrogen (m/z 28), oxygen
(m/z 32), or carbon dioxide (m/z 44) shall be less than 5% of the
base peak for the system to be considered leak free.
    <IP_ADDRESS>.2 Tune the MSD using the Standard Autotune program and
the decafluorotriphenylphosphine (DFTPP) Tune program to reduce
instrument variability. The Autotune report file is referenced by
the instrument when performing an air/water check and thus must be
run at least once per month. Run standards and samples using DFTPP
Tune parameters, and retune the instrument using DFTPP Tune at least
once per week. The tune programs use three fragment ions of
perfluorotributylamine (PFTBA) as a standard for tuning: m/z 69,
219, and 502. Tune reports must meet the following criteria:
    <IP_ADDRESS>.2.1 Symmetrical peaks;
    <IP_ADDRESS>.2.2 Mass assignments within  0.2 amu's from
69, 219, and 502;
    <IP_ADDRESS>.2.3 Peak widths within 0.5  0.1 amu's;
    <IP_ADDRESS>.2.4 Relative abundance is 100% for ion 69, at least 35%
for ion 219, and at least 1% for ion 502;
    <IP_ADDRESS>.2.5 Relative abundances for isotope masses 70, 220, and
503  0.2 amu's are 0.5-1.5%, 2-8%, and 5-15%,
respectively; and
    <IP_ADDRESS>.2.6 Air and water peaks at m/z = 18, 28, 32, and 44
amu's must be very small and consistent with historical values.
    <IP_ADDRESS> Maintaining a log book. Maintain an instrument logbook,
and make entries for each use. Include the following information in
the logbook: operator name, helium cylinder tank pressure and outlet
pressure, vacuum gauge reading, any maintenance performed on the
instrument (such as changing the injection port liner, gold seal,
guard column, source cleaning), sequence name, data path, samples in
order of injection, method information, GC column number, and the
Standard Auto Tune report and DFTPP Tune report.
    <IP_ADDRESS> Running a Solvent Blank: Following a liner change or at
the start of a new run, run an injection of a pure solvent to
confirm that the system is free of excessive or interfering
contamination. Analyze the solvent in SCAN mode using the same
temperature program used for sample analysis. If contamination is
present, analyze additional samples of fresh solvent until the
interfering contamination is removed.
    <IP_ADDRESS> Checking the DFTPP Tune: Prior to running the first
calibration standard, verify the instrument tune conditions by
running a 10 ng/[mu]L DFTPP check standard to check the mass
measuring accuracy of the MS, the resolution sensitivity, the
baseline threshold, and the ion abundance ranges. Run the standard
using the DFTPP method provided with the instrument. Each of the
criteria identified in Table SOP 4.2 of this Appendix must be met
before using the instrument for analysis:

                                 Table SOP 4.3--Ion Abundance Criteria for DFTPP
----------------------------------------------------------------------------------------------------------------
                                           Relative to
                Mass, M/z                      mass     Relative abundance criteria     Purpose of checkpoint
----------------------------------------------------------------------------------------------------------------
51.......................................          442  10-80% of the base peak....  Low mass sensitivity.
68.......................................           69  <2% of mass 69.............  Low mass resolution.
70.......................................           69  <2% of mass 69.............  Low mass resolution.
127......................................          442  10-80% of the base peak....  Low-mid mass sensitivity.
197......................................          198  <2% of mass 198............  Mid mass resolution.
198......................................          442  Base peak or >50% of 442...  Mid mass resolution and
                                                                                sensitivity.
199......................................          198  5-9% of mass 198...........  Mid mass resolution and
                                                                                isotope ratio.
275......................................          442  10-60% of the base peak....  Mid-high mass sensitivity.
365......................................          442  >1% of the base peak.......  Baseline threshold.
441......................................          443  Present and < mass 443.....  High mass resolution.
442......................................          442  Base peak or >50% of 198...  High mass resolution and
                                                                                sensitivity.
443......................................          442  15-24% of mass 442.........  High mass resolution and
                                                                                isotopic ratio.
----------------------------------------------------------------------------------------------------------------

    <IP_ADDRESS> Calibrating with a Multiple-Point Calibration Curve. A
5- or 6-point calibration curve is obtained by running 5 or 6
working standards (see SOP 3) on the tuned GC/MS instrument.
Calculate the relative response factor (RRF) for each compound
relative to its corresponding deuterated internal standard as
indicated in Table SOP 4.3 of this Appendix. The relative standard
deviation (RSD) of the RRFs for each compound must be less than 25%.
Run an independently prepared check standard immediately after the
calibration standards to validate the accuracy of the calibration
curve.
    <IP_ADDRESS> Running Samples. Once the calibration curve has been
validated, samples can be analyzed. Dispense 1,000 [mu]L of sample
extract into labeled auto-sampler vials. Add

20 [mu]L of the internal standard solution (see SOP 2 of this
Appendix) to the extract using a syringe or a positive displacement
pipettor. Run a check standard every 10 samples to ensure the
consistency of the instrument. The RRF for each compound in the
check standard must be within 25% of the average RRF obtained in the
initial calibration.
    <IP_ADDRESS> Quantification: Once a calibration table has been
generated, quantify each data file using the ``Calculate and
Generate'' function in the MS ChemStation software, or equivalent
software. Review individual peak integration manually to ensure
proper baseline integration. The quantification of a compound is
based on the peak area of the primary ion (Q Ion) indicated in Table
SOP 4.4 of this Appendix.

                                       Table SOP 4.4--Target Compound List
----------------------------------------------------------------------------------------------------------------
                                           Quantitation     Reference compound for       Internal standard for
              Compound name                     ion             response factor              quantitation
----------------------------------------------------------------------------------------------------------------
N D34 C16...............................              66  N D34 C16.................  D34 n C16 Q Ion 66.
n-C14...................................              57  n C14.....................
n-C15...................................              57  n C15.....................
n-C16...................................              57  n C16.....................
N D34 C17...............................              66  N D34 C17.................
n-C17...................................              57  n C17.....................
Pristane................................              57  Pristane..................
n-C18...................................              57  n C18.....................
Phytane.................................              57  Phytane...................
n C19...................................              57  n C19.....................
N D42 C20...............................              66  N D42 C20.................  D42 n C20 Q Ion 66.
n C20...................................              57  n C20.....................
n C21...................................              57  n C21.....................
n C22...................................              57  n C22.....................
n C23...................................              57  n C23.....................
N D50 C 24..............................              66  N D50 C 24................
n C24...................................              57  n C24.....................
n C25...................................              57  n C25.....................
n C26...................................              57  n C26.....................
n C27...................................              57  n C27.....................
n C28...................................              57  n C28.....................
n C29...................................              57  n C29.....................
N D62 C30...............................              66  N D62 C30.................  D62 n C30Q Ion 66.
n C30...................................              57  n C30.....................
n C31...................................              57  n C31.....................
N D66 C32...............................              57  N D66 C32.................
n C32...................................              57  n C32.....................
n C33...................................              57  n C33.....................
n C34...................................              57  n C34.....................
n C35...................................              57  n C35.....................
D8 Naphthalene..........................             136  D8 Naphthalene............  D8 Naphthalene Q Ion 136.
Naphthalene.............................             128  Naphthalene...............
D10 1-Methylnaphthalene.................             152  D10 1-Methylnaphthalene...
C1 Naphthalene *........................             142  C1 Naphthalene............
C2 Naphthalene *........................             156  C2 Naphthalene............
C3 Naphthalene *........................             170  C3 Naphthalene............
C4 Naphthalene *........................             184  C3 Naphthalene............
D10 Anthracene..........................             188  D10 Anthracene............  D10 Anthracene Q Ion 188.
D10 Phenanthrene........................             188  D10 Phenanthrene..........
Phenanthrene............................             178  Phenanthrene..............
C1 Phenanthrene *.......................             192  C1 Phenanthrene...........
C2 Phenanthrene *.......................             206  C2 Phenanthrene...........
C3 Phenanthrene *.......................             220  C2 Phenanthrene...........
C4 Phenanthrene *.......................             234  C2 Phenanthrene...........
Fluorene................................             166  Fluorene..................
C1 Fluorene *...........................             180  Fluorene..................
C2 Fluorene *...........................             194  Fluorene..................
C3 Fluorene *...........................             208  Fluorene..................
Dibenzothiophene........................             184  Dibenzothiophene..........
C1 Dibenzothiophene *...................             198  Dibenzothiophene..........
C2 Dibenzothiophene *...................             212  Dibenzothiophene..........
C3 Dibenzothiophene *...................             226  Dibenzothiophene..........
Naphthobenzothiophene (NBT).............             234  Naphthobenzothiophene.....
C1 NBT *................................             248  Naphthobenzothiophene.....
C2 NBT *................................             262  Naphthobenzothiophene.....
C3 NBT *................................             276  Naphthobenzothiophene.....
Fluoranthene............................             202  Fluoranthene..............
D10 Pyrene..............................             212  D10 Pyrene................
Pyrene..................................             202  Pyrene....................
C1 Pyrene *.............................             216  Pyrene....................
C2 Pyrene *.............................             230  Pyrene....................
D12 Chrysene............................             240  D12 Chrysene..............  D12 Chrysene Q Ion 240.
Benzo(a)anthracene/Chrysene *...........             228  Chrysene..................
C1 Chrysene *...........................             242  Chrysene..................

C2 Chrysene *...........................             256  Chrysene..................
C3 Chrysene *...........................             270  Chrysene..................
C4 Chrysene *...........................             284  Chrysene..................
5[alpha]-androstane.....................             245  5[alpha]-androstane.......  5[alpha]-androstane Q Ion
                                                                                245
Coprostane..............................             219  Coprostane................
Hopane..................................             191  Hopane....................
----------------------------------------------------------------------------------------------------------------
* Summed compounds; draw an integration line underneath all peaks with selected ion.

    <IP_ADDRESS> Equation 14 of this Appendix is used to calculate the
concentration of analytes in units of [mu]g/g oil added:
[GRAPHIC] [TIFF OMITTED] TP22JA15.054

where:

Aanalyte = the peak area of the analyte,
Cistd = the concentration of the internal standard,
Aistd = the area of the internal standard,
RRF = the relative response factor, and
100 is the conversion factor to convert mg/L DCM to [mu]g/g oil
added.

    <IP_ADDRESS> If some analytes are not commercially available, the
RRFs of other compounds (usually the parent compound) are used to
quantify those analytes. For example, the RRF of C3-naphthalene may
be used to calculate the concentrations of C3- and C4-naphthalenes.
See Table SOP 4.4 of this Appendix for details. The quantification
of these alkylated PAHs is relative because it is assumed that the
molecular ions of the alkylated PAHs have the same RRFs as the
parent compound ions. Nevertheless, these relative concentrations
are useful for monitoring the fate of these compounds during the
course of any analysis, as long as their concentrations are measured
in a consistent way throughout the analysis.
    <IP_ADDRESS> Concentration calculations for all target compounds
are performed using EnviroQuant software or equivalent. Data for
each sample can be printed directly using a customized report
template. Data can also be automatically entered into a spreadsheet
within the EnviroQuant software.
    5.10.5 Quality Assurance/Quality Control. The following criteria
must be met before any samples are analyzed:
    <IP_ADDRESS> Air/water check to verify the system is leak free.
    <IP_ADDRESS> AutoTune and DFTPP Tune pass all criteria.
    <IP_ADDRESS> DFTPP check standard passes all criteria.
    <IP_ADDRESS> Solvent blank scan indicates the GC/MS system is free
of interfering contamination.
    <IP_ADDRESS> Prepare and monitor a control chart of a standard oil
analysis. Concentrations of the analytes in the control chart must
be no more than 25% different from their historical averages.
    <IP_ADDRESS> Relative response factors for analytes in the check
standards inserted between every 10 samples must be no more than 25
percent different from the average RRF of those same analytes in the
calibration curve. Peak shapes must be symmetrical.

5.11 References for Section 5

(1) Haines, J.R., E.J. Kleiner, K.A. McClellan, K.M. Koran, E.L.
Holder, D.W. King, and A.D. Venosa. 2005. ``Laboratory evaluation of
oil spill bioremediation products in salt and freshwater systems.''
J. Ind. Microbiol. Biotech 32: 171-185.
    Appendix E to Part 300 [Removed]
0
17. Remove Appendix E to Part 300.

[FR Doc. 2015-00544 Filed 1-21-15; 8:45 am]
BILLING CODE 6560-50-P