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1.0 Introduction
2.0 Swirling Flask Dispersant Effectiveness Test
3.0 Revised Standard Dispersant Toxicity Test
4.0 Bioremediation Agent Effectiveness Test
5.0 Bioremediation Agent Toxicity Test
6.0 Summary Technical Product Test Data Format
1 Swirling Flask Test Apparatus
1 Major Ion Composition of "Instant Ocean" Synthetic Sea Salt
2 Test Oil Characteristics
3 Oil Standard Solutions: Concentrations in Final DCM Extractions
4 Synthetic Seawater [Toxicity Test]
5 Test Oil Characteristics: No. 2 Fuel Oil
6 Analytes Listed Under the Corresponding Internal Standard Used in Calculating RRFs
7 Primary Ions Monitored for Each Target Analyte During GC/MS Analysis
8 Analytes and Reference Compounds
9 Operating Conditions and Temperature Program of GC/MS
10 Two-Way ANOVA Table
11 Product Test Data, Total Aromatics
12 Summary Statistics for Product Test Data, Total Aromatics
13 Example Two-Way ANOVA Table
14 Pairwise Protected LSD Mean Separation
1.1 Scope and Application. The methods described below apply to "dispersants, surface washing agents, surface collecting agents, bioremediation agents, and miscellaneous oil spill control agents" involving subpart J (Use of Dispersants and Other Chemicals) in 40 CFR part 300 (National Oil and Hazardous Substances Pollution Contingency Plan). They are revisions and additions to the EPA's Standard Dispersant Effectiveness and Toxicity Tests (1). The new Swirling Flask Dispersant Effectiveness Test is used only for testing dispersants. The Revised Standard Dispersant Toxicity Test is used for testing dispersants, as well as surface washing agents, surface collecting agents, and miscellaneous oil spill control agents. The bioremediation agent effectiveness test is used for testing bioremediation agents only.
1.2 Definitions. The definitions of dispersants, surface washing agents, surface collecting agents, bioremediation agents, and miscellaneous oil spill control agents are provided in 40 CFR 300.5.
2.1 Summary of Method. This protocol was developed by Environment Canada to provide a relatively rapid and simple testing procedure for evaluating dispersant effectiveness (2). It uses a modified Erlenmeyer flask to which a side spout has been added for removing subsurface samples of water near the bottom of the flask without disturbing a surface oil layer. Seawater and a surface layer of oil are added to the flask. Turbulent mixing is provided by placing the flask on a standard shaker table at 150 rpm for 20 minutes to induce a swirling motion to the liquid contents. Following shaking, the flask is immediately removed from the shaker table and maintained in a stationary position for 10 minutes to allow the oil that will reform a slick to return to the water's surface. A sample of water for chemical analysis is then removed from the bottom of the flask through the side spout, extracted with methylene chloride (dichloromethane-DCM), and analyzed for oil content by UV-visible absorption spectrophotometry at wavelengths of 340, 370, and 400 nm (2).
2.2 Apparatus.
2.2.1 Modified Erlenmeyer Flask. Use 125-ml glass Erlenmeyer flasks that have been modified to include an attachment of a glass side spout that extends from the bottom of the flask upward to the neck region, as shown in Figure 1.
2.2.2 Shaker Table. Use a shaker table with speed control unit with variable speed (40-400 rpm) and an orbital diameter of approximately 0.75 inches (2 cm) to provide turbulence to solutions in test flasks.
2.2.3 Spectrophotometer. Use a UV-visible spectrophotometer capable of measuring absorbance at 340, 370, and 400 nm. A Hitachi Model U-2000 or equivalent is acceptable for this purpose.
2.2.4 Glassware. Glassware should
consist of 5-, 10-, 25-, 100-, and 500-ml graduated cylinders; 125-ml separatory
funnels with Teflon stopcocks; and 10-, 100-, and 1,000-ml volumetric flasks and
micropipettes.
2.3 Reagents. 2.3.1 Synthetic seawater. The synthetic sea salt
"Instant Ocean," manufactured by Aquarium Systems of Mentor, OH, can be used for
this purpose. The synthetic seawater solution is prepared by dissolving 34 g of
the salt mixture in 1 liter of distilled water (i.e., a salinity of 34 ppt).
Table 1 provides a list of the ion composition of the seasalt mixture. Following the preparation, the saltwater solution is allowed to equilibrate
to the ambient temperature of the laboratory and should be in the range of 22±3
°C.
2.3.2 Test oil. Two EPA/American
Petroleum Institute (API) standard reference oils, Prudhoe Bay and South
Louisiana crude, should be used for this test. These oils can be obtained from
the Resource Technology Corporation, 2931 Soldier Springs Road, P.O. Box 1346,
Laramie, WY 82070, (307) 742-5452. These oils have been thoroughly homogenized,
as well as characterized physically and chemically for previous EPA and API
studies. Various selected parameters are presented in table 2. 2.3.3 Methylene Chloride
(Dichloromethane-DCM), pesticide quality. For extraction of all sample water
and oil-standard water samples.
2.4 Pretest preparation. 2.4.1
Preparation and analysis of oil standards. 2.4.1.1 Standard solutions of
oil for calibrating the UV-visible spectrophotometer are prepared with the
specific reference oils and dispersant used for a particular set of experimental
test runs. For experiments with no dispersant, only oil is used to make the
standard solution. For experiments with the oil plus dispersant, the standard is
made with a 1:10 (v:v) mixture of the dispersant to the test oil (i.e., a
dispersant-to-oil ratio of 1:10). This ratio is used in the test tank with
dispersant added. The presence of water and certain dispersants in DCM extracts
can affect absorbance readings in a spectrophotometer. All standard solutions of
oil (and dispersant, if present) should be prepared in a stepwise manner that
reflects the analytical protocol used for the experimental water samples.
2.4.1.2 To prepare the standards, prepare
a parent oil-DCM standard by mixing 1 part oil (plus 1/10 part premixed
dispersant, if applicable) to 9 parts DCM (i.e., 1:10 dilution of the oil v:v).
Add a specific volume of the parent oil-DCM standard to 30 ml of synthetic
seawater in a separatory funnel. Extract the oil-water mixture with 5-ml volumes
of DCM after 15 seconds of vigorous shaking followed by a 2 minute stationary
period to allow for phase separation for each extraction. Repeat the extraction
using a total of three 5-ml portions of DCM. Adjust the final DCM volume for the
combined extracts to 20 ml with DCM in a 25-ml graduated cylinder.
2.4.1.3 The quantities of oil used to
achieve the desired concentrations in the final 20-ml DCM extracts for the
standard oil-solutions are summarized in table 3. Specific masses for oil
amounts in standards are determined as volumes of oil multiplied by the density
of the oil.
2.4.2 Linear stability calibration of
UV-Visible spectrophotometer.
2.4.2.1 Before DCM-extracts of dispersed
oil-water samples can be analyzed for their oil content, the UV-visible
spectrophotometer must meet an instrument stability calibration criterion. This
criterion is determined with the six oil standards identified in table 3.
Determine the absorbance of standards at each of the three analytical
wavelengths (i.e., 340, 370, and 400 nm). Determine the response factors (RFs)
for the test oil at each of the three analytical wavelengths using the following
equation: RF where:
RF C=Oil concentration, in mg of oil/ml of DCM in standard solution
A 2.4.2.2 Instrument stability for the
initial calibration is acceptable when the RFs for the five highest standard
extracts of oil are <20% different from the overall mean value for the five
standards. If this criterion is satisfied, analysis of sample extracts can
begin. RFs for the lowest concentration (0.05 mg oil/ml DCM) are not included in
the consideration because the absorbance is close to the detection limit of the
spectrophotometer (with associated high variability in the value) for the 1-cm
path-length cell used for measurements. Absorbances ≥3.5 are not included
because absorbance saturation occurs at and above this value.
2.4.2.3 If one or more of the standard oil
extracts do not meet this linear-stability criterion, then the "offending"
standard(s) can be prepared a second time (i.e., extraction of the specified
amount of oil from 30-ml or seawater for the "offending" standard according to
the pretest preparation procedure). If replacement of the reanalyzed standard
solution(s) in the standard curve meets the linear-stability criterion (i.e., no
RF >20% different from the overall mean), then analysis of sample extracts
can begin.
2.4.2.4 If the initial-stability criterion
is still not satisfied, analysis of sample extract cannot begin and the source
of the problem (e.g., preparation protocol for the oil standards,
spectrophotometer stability, etc.) must be corrected.
2.4.2.5 The initial six-point calibration
of the UV-visible spectrophotometer at the oil concentrations identified is
required at least once per test day.
2.5 Test procedure. 2.5.1
Preparation of premixed dispersant oil. Prepare a premixed dispersant oil
by mixing 1 part dispersant to 10 parts oil. Store this mixture in a glass
container. The dispersant effectiveness test procedures are listed in steps
1-20:
1. Prepare 4 replicates (same test oil and dispersant), one control (i.e., no
dispersant), and one method blank and run at the same time on the shaker table.
2. Add 120±2 ml of synthetic seawater to each of the modified 125-ml glass
Erlenmeyer flasks. Measure and record the water temperature.
3. Place the flasks securely into the attached slot on the shaker table.
4. Carefully add 100 μl of an oil-dispersant solution onto the center of the
water's surface using a positive displacement pipette.
5. Agitate the flasks for 20±1 minutes at 150±10 rpm on the shaker table.
6. After the 20±1 minutes shaking, remove the flasks from the shaker table
and allow them to remain stationary for 10±1 minutes for oil droplet "settling."
7. At the conclusion of the 10-minute settling period, carefully decant a
30-ml sample through the side spout of the test flasks into a 50-ml graduated
cylinder.
Note: Discard the first 1-2 ml of sample water to remove
nonhomogeneous water-oil initially contained in the spout.
8. Transfer the samples from the graduated cylinder into a 125- or 250-ml
glass separatory funnel fitted with a Teflon stopcock.
9. Add 5 ml of pesticide-quality DCM to the separatory funnel and shake
vigorously for 15 seconds. Release the pressure carefully from the separatory
funnel through the stopcock into a fume hood.
10. Allow the funnel to remain in a stationary position for 2 minutes to
allow phase-separation of the water and DCM.
11. Drain the DCM layer from the separatory funnel into a glass-stoppered,
25-ml graduated glass cylinder.
12. Repeat the DCM-extraction process two additional times.
13. Combine the three extracts in the graduated cylinder and adjust the final
volume to 20-ml with additional DCM.
14. Analyze the samples using a UV-spectrophotometer at 340, 370, and 400
nm-wavelengths and determine the quantity of oil as follows: C where:
C A RF V V V 15. Obtain three concentration values for oil in each experimental water
sample (340, 370, and 400 nm).
16. Determine the mean of three values as follows:
C Note: Means will be used for all dispersion-performance calculations.
Samples where one of the values for C 17. Determine the dispersant performance (i.e., percent of oil that is
dispersed, or EFF) based on the ratio of oil dispersed in the test system to the
total oil added to the system as follows: EFF (in %)=(C where:
C C 18. Calculate EFF using equation 4 for coupled experiments with and without
dispersant (EFF 19. Calculate the final dispersant performance of a chemical dispersant agent
after correcting for natural dispersion using equation 5. EFF where:
EFF EFF EFF 20. Calculate the average dispersant effectiveness value by summing the
corrected values (EFF 2.6 Performance criterion. The dispersant product tested will remain
in consideration for addition to the NCP Product Schedule if the average
dispersant effectiveness, as calculated in section 2.5 above, is at least 45%
(i.e., 50%±5%).
2.7 Quality Control (QC) procedures for
measurements of oil concentrations. 2.7.1 UV-visible spectrophotometric
measurements. At least 5% of all UV-visible spectrophotometric measurements
will be performed in duplicate as a QC check on the analytical measurement
method. The absorbance values for the duplicates should agree within ±5% of
their mean value.
2.7.2 Method blanks. Analytical
method blanks involve an analysis of seawater blanks (i.e., seawater but no oil
or dispersant in a swirling flask vessel) through testing and analytical
procedures (3, pp 79-80). Method blanks are analyzed with a frequency of at
least 1 for every 12 experimental swirling flask samples. Oil concentrations in
method blanks must be <5% of that occurring for 100% dispersion of oil in
testing apparatus.
3.1 Summary of method. The standard
toxicity test for dispersants and other products involves exposing two species
(Menidia beryllina (silversides) and Mysidopsis bahia (mysid shrimp)) to five
concentrations of the test product and No. 2 fuel oil alone and in a 1:10
mixture of product to oil. To aid in comparing results from assays performed by
different workers, reference toxicity tests are conducted using dodecyl sodium
sulfate (DSS) as a reference toxicant. The test length is 96 hours for Menidia
and 48 hours for Mysidopsis. LC 3.2 Selection and preparation of test
materials.
3.2.1 Test organisms.
3.2.1.1 Menidia beryllina. Obtain
fish (silversides) from a single source for each series of toxicity tests.
In-house cultures are recommended wherever it is cost-effective; however,
organisms are available from commercial suppliers. Information on the source of
test organisms and any known unusual condition to which fish were exposed before
use should be included in the data report. Use of animals previously treated
with pesticides or chemotherapeutic agents should be avoided. Organisms should
not be used if they appear to be unhealthy, discolored, or show signs of stress.
Use 7-day old larval fish. Fish should be cultured in accordance with the
methods outlined in Middaugh, et al. (5). There should be no need to acclimate
organisms to the 25±1 °C temperature recommended for the toxicity tests if
laboratory stock cultures of Menidia are maintained at the recommended culture
temperature of 25±1 °C. If test organisms must be obtained from a commercial
source, it may become necessary to acclimate test fish to the test temperature
of 25±1 °C, a pH of 8.0±0.2, and 20±2 ppt salinity since changes in temperature
may occur during shipping. Eliminate groups of fish having a mortality of more
than 10% during the first 48 hours, and more than 5% thereafter. During
acclimation, organisms should be maintained on a diet of freshly hatched Artemia
(brine shrimp) nauplii. Feed the fish daily to satiation during the acclimation
period, and once daily during the 96-hour test. Care should be taken daily to
remove excess food and fecal material from beakers during the test. Use only
those organisms that feed actively and that appear to be healthy. Organisms
should be free of disease, external parasites, and any signs of physical damage
or stress. Discard any fish injured or dropped while handling.
3.2.1.2 Mysidopsis bahia. Several
methods for culturing Mysidopsis bahia (mysid shrimp) may be used and are noted
in appendix A of Methods for Measuring the Acute Toxicity of Effluents and
Receiving Waters to Freshwater and Marine Organisms (6). To ensure uniformity of
mysids, recently hatched mysids should be collected daily from stock cultures
and identified by the date of hatch. Mysids used in 48-hour tests should be from
a single day's collection, but may have an age range of 5-7 days old. In cases
where in-house cultures of mysids are unavailable, organisms may be purchased
from a commercial source. Information on the source of test organisms should be
submitted in the data report.
3.2.2 Preparation of experimental
water. Filtered natural seawater is recommended for use since it represents
a natural source of saltwater containing an inherent population of
microorganisms. Synthetic seawater formulated according to the following method
can serve as an acceptable alternative to filtered, natural seawater for
toxicity tests performed in laboratories in which natural seawater is
unavailable.
3.2.3 Synthetic seawater formation.
To prepare standard seawater, mix technical-grade salts with 900 liters of
distilled or demineralized water in the order and quantities listed in table 4.
These ingredients must be added in the order listed and each ingredient must be
dissolved before another is added. Stir constantly after each addition during
preparation until dissolution is complete. Add distilled or demineralized water
to make up to 1,000 liters. The pH should now be 8.0±0.2. To attain the desired
salinity of 20±1 ppt, dilute again with distilled or demineralized water at time
of use.
3.3 Sampling and storage of test
materials. Toxicity tests are performed with No. 2 fuel oil having the
characteristics defined in table 5. Store oil used for toxicity tests in sealed
containers to prevent the loss of volatiles and other changes. For ease in
handling and use, it is recommended that 1,000-ml glass containers be used. To
ensure comparable results in the bioassay tests, use oils packaged and sealed at
the source. Dispose of unused oil in each open container on completion of dosing
to prevent its use at a later date when it may have lost some of its volatile
components. Run all tests in a bioassay series with oil from the same container
and with organisms from the same group collected or secured from the same
source. 3.4 General test conditions and
procedures for toxicity tests.
3.4.1 Temperature. For these toxicity tests, use test solutions with
temperatures of 25±1 °C.
3.4.2 Dissolved oxygen and aeration.
3.4.2.1 Menidia. Because oils contain toxic, volatile materials, and
because the toxicity of some water-soluble fractions of oil and degradation
products are changed by oxidation, special care must be used in the oxygenation
of test solutions. Aeration during the test is generally not recommended but
should be used to maintain the required dissolved oxygen (DO) in cases where low
DO is observed. The DO content of test solutions must not drop below 60%
saturation during the first 48 hours of a static acute (96-hour) test and must
remain between 40-100% after the first 48 hours of the test. Aeration at a rate
of 100±15 bubbles per minute is supplied by a serological pipette as needed for
maintenance of DO. If aeration is necessary, all test chambers should be
aerated. At this rate, and with the proper weight of fish, DO concentration
should remain slightly above 4 ppm over a 96-hour period. Take DO measurements
daily. 3.4.2.2 Mysidopsis. Achieve sufficient DO by ensuring that the surface
area to volume ratio of the test solution exposed is large enough. Oxygen
content should remain high throughout the test because of the low oxygen demand
of the organisms. Aeration is not recommended during 48-hour acute toxicity
tests unless the DO falls below 60% saturation.
3.4.3 Controls. With each fish or mysid test or each series of
simultaneous tests of different solutions, perform a concurrent control test in
exactly the same manner as the other tests and under the conditions prescribed
or selected for those tests. Use the diluent water alone as the medium in which
the controls are held. There must be no more than 10% mortality among the
controls during the course of any valid test.
3.4.4 Reference toxicant. To aid in comparing results from tests
performed by different workers and to detect changes in the condition of the
test organisms that might lead to different results, perform reference toxicity
tests with reagent grade DSS in addition to the usual control tests. Prepare a
stock solution of DSS immediately before use by adding 1 gram of DSS per 500 ml
of test water solution. Use exploratory tests before the full scale tests are
begun to determine the amount of reference standard to be used in each of the
five different concentrations.
3.4.5 Number of organisms. At a minimum, 20 organisms of a given
species are exposed for each test concentration. For the toxicity test
procedures using Menidia, place 10 fish in each of two jars. For the toxicity
tests using Mysidopsis, place 10 larvae in each of two containers.
3.4.6 Transfer of organisms. Organisms should be handled as little as
possible in order to minimize stress. Transfer Menidia and Mysidopsis from the
acclimatization aquaria to the test chambers with a pipette or a wide-bore,
smooth glass tube (4 to 8 mm internal diameter) fitted with a rubber bulb. Dip
nets should be avoided when handling larval fish and mysids. Do not hold fish
out of the water longer than necessary and discard any specimen accidentally
dropped or otherwise mishandled during transfer.
3.4.6.1 Mysidopsis. To have the mysids ready for study, mysids may be
sorted 24 hours prior to initiation of the 48-hour test. Transfer the mysids to
a beaker containing a small volume of water; this vessel serves as a holding
chamber during randomized transfer of the organisms to test solutions. Mysids
are randomly selected from the batch of mysids in the holding chamber, and
transferred to 50-ml beakers containing a small volume of seawater. One mysid is
added per beaker using a small piece of flexible 500-μm screening until all of
the beakers contain one mysid. The process of random selection and sorting is
continued until the appropriate number of mysids has been delivered to each of
the 50-ml beakers. The mysids are gently released from the 50-ml beakers into
larger beakers filled with an appropriate volume of 20-ppt seawater (25 °C) to
bring the total volume to 200 ml. The beakers are randomly placed into a
temperature-controlled water bath to acclimate overnight at 25 °C. The mysids
are transferred to larger beakers (1-liter) for the 48-hour test after the
addition of 800 ml of the test solution. A total of 10 mysids per beaker are
used for 48-hour acute toxicity tests. A minimum of two replicate chambers are
used for each test concentration and control.
3.4.6.2 Menidia and Mysidopsis are
fed 50 brine shrimp nauplii/organism daily during the 96-hour and 48-hour tests.
Excess food should be removed daily by aspirating with a pipette.
3.4.7 Test duration and
observations. 3.4.7.1 Menidia. Observe the number of dead fish in
each test container and record at the end of each 24-hour period. Fish are
considered dead upon cessation of respiratory and all other overt movements,
whether spontaneous or in response to mild mechanical prodding. Remove dead fish
as soon as observed. Also note and report when the behavior of test fish
deviates from that of control fish. Such behavioral changes would include
variations in opercular movement, coloration, body orientation, movement, depth
in container, schooling tendencies, and others. Abnormal behavior of the test
organisms (especially during the first 24 hours) is a desirable parameter to
monitor in a toxicity test because changes in behavior and appearance may
precede mortality. Toxicants can reduce an organism's ability to survive natural
stresses. In these cases, the mortality is not directly attributed to the
toxicant, but most certainly is an indirect effect. Reports on behavioral
changes during a toxicity test can give insight into the non-acute effects of
the tested material. At the end of the 96-hour period, terminate the fish tests
and determine the LC 3.4.7.2 Mysidopsis. Terminate the
mysid test after 48 hours of incubation. To count the dead animals accurately,
place the exposure vessels on a light table such that light passes through the
bottom of the vessel. Most of the dead mysids will be on the bottom of the
beaker and can readily be seen against the background of the light table. Also
search the top of the liquid for mysids trapped there by surface tension.
Exercise caution when determining death of the animals. Occasionally, an animal
appears dead, but closer observation shows slight movement of an appendage or a
periodic spasm of its entire body. For these tests, animals exhibiting any
movement when touched with a pipette tip are considered alive. Account for all
test animals to ensure accuracy since Mysidopsis bahia may disintegrate
or be cannibalized by other mysids. Consider individuals not accounted for as
dead. At the end of 48 hours of exposure, terminate the mysid assay and
determine the LC 3.4.8 Physical and chemical
determinations. 3.4.8.1 Menidia. Determine the temperature, DO, and
pH of the test solutions before the fish are added and at 24-, 48-, 72-, and
96-hour exposure intervals. It is necessary to take measurements from only one
of the replicates of each of the toxicant series.
3.4.8.2 Mysidopsis. Determine the
temperature, DO, and pH of the test solutions before the nauplii are added and
at the 24- and 48-hour exposure interval. Measure DO and pH in only one of the
replicates of each of the toxicant series.
3.4.9 Testing laboratory. An
ordinary heated or air-conditioned laboratory room with thermostatic controls
suitable for maintaining the prescribed test temperatures generally will suffice
to conduct the toxicity tests. Where ambient temperatures cannot be controlled
to 25±1 °C, use water baths with the necessary temperature controls.
3.4.10 Test containers. For tests
with fish or mysids, use 1-liter glass beakers measuring approximately 10 cm in
diameter. In conducting the test, add to each beaker 1 liter of the test
solution or seawater formulation aerated to saturation with DO. To add the liter
volume easily and accurately, use a large volume (1-liter) graduated cylinder.
Process all required glassware before each test. Immerse in normal hexane for 10
minutes. Follow this with a thorough rinse with hot tap water; three hot
detergent scrubs; an additional hot tap-water rinse; and three rinses with
distilled water. Oven or air dry the glassware in a reasonably dust-free
atmosphere.
3.5 Preparation of test
concentrations. 3.5.1 Menidia.
Place test jars (approximately 22.5 cm in height, 15 cm in diameter, 11 cm in
diameter at the mouth) containing 2 liters of synthetic seawater on a reciprocal
shaker. The shaker platform should be adapted to hold firmly six of the toxicity
test jars. Add the desired amount of the petroleum product (if applicable) under
test directly to each test jar. Dispense the appropriate amount of toxicant (if
applicable) into the jars with a pipette. Tightly cap the test jars and shake
for 5 minutes at approximately 315 to 333 2-cm (0.75-inch) strokes per minute in
a reciprocal shaker or at approximately 150 to 160 rpm on orbital shakers. At
the completion of shaking, remove the jars from the shaker and dispense 1 liter
of the mixture to each of the 1-liter glass beakers. Randomly place beakers in a
constant-temperature water bath or room, take water quality measurements, add
fish, and initiate aeration.
3.5.2 Mysidopsis.
3.5.2.1 To prepare test solutions for
products and oil/product mixtures, blend or mix the test solutions with an
electric blender having: speeds of 10,000 rpm or less; a stainless-steel cutting
assembly; and a 1-liter borosilicate jar. To minimize foaming, blend at speeds
below 10,000 rpm.
3.5.2.2 For the product test solution, add
550 ml of the synthetic seawater to the jar, then with the use of a gas-tight
calibrated glass syringe with a Teflon-tipped plunger, add 0.55 ml of the
product and mix for 5 seconds.
3.5.2.3 For the oil test solution, add 550
ml of the synthetic seawater to the jar. Then with the use of a gas-tight
calibrated glass syringe equipped with a Teflon-tipped plunger, add 0.55 ml of
the oil and mix for 5 seconds.
3.5.2.4 For the oil/product mixture, add
550 ml of the synthetic seawater to the mixing jar. While the blender is in
operation, add 0.5 ml of the oil under study with the use of a calibrated
syringe with a Teflon-tipper plunger and then 0.05 ml of the product as
indicated above. Blend for 5 seconds after addition of product. These additions
provide test solutions of the product, oil, and the oil/product mixture at
concentrations of 1,000 ppm.
3.5.2.5 Immediately after the test
solutions are prepared, draw up the necessary amount of test solution with a
gas-tight Teflon-tipped glass syringe of appropriate size and dispense into each
of the five containers in each series. If the series of five concentrations to
be tested are 10, 18, 32, 56, and 100 ppm, the amount of the test solution in
the order of the concentrations listed above would be as follows: 10, 18, 32,
56, and 100 ml.
3.5.2.6 Each time a syringe is to be
filled for dispensing to the series of test containers, start the mixer and
withdraw the desired amount in the appropriate syringe while the mixer is in
operation. Turn off immediately after the sample is taken to limit the loss of
volatiles.
3.5.2.7 Use exploratory tests before the
full-scale test is set up to determine the concentration of toxicant to be used
in each of the five different concentrations. After adding the required amounts
of liquid, bring the volume in each of the test containers up to 800 ml with the
artificial seawater. To ensure keeping each of the series separate, designate on
the lid of each container the date, the material under test, and its
concentration.
3.5.2.8 When the desired concentrations
are prepared, gently release into each beaker the 10 test Mysidopsis
(previously transferred into 200 ml of medium). This provides a volume of 1
liter in each test chamber. A pair of standard cover glass forceps with flat,
bent ends is an ideal tool for handling and tipping the small beaker without
risk of contaminating the medium.
3.5.2.9 After adding the test animals,
incubate the test beakers at 25±1 °C for 48 hours. Recommended lighting is 2,000
lumens/m 2 (200 ft-c) of diffused, constant, fluorescent
illumination.
3.5.2.10 Wash the blender thoroughly after
use and repeat the above procedures for each series of tests. Wash the blender
as follows: rinse with normal hexane; pour a strong solution of laboratory
detergent into the blender to cover the blades; fill the container to about half
of its volume with hot tap water; operate the blender for about 30 seconds at
high speed; remove and rinse twice with hot tap water, mixing each rinse for 5
seconds at high speed; and then rinse twice with distilled water, mixing each
rinse for 5 seconds at high speed.
3.6 Calculating and reporting. At
the end of the test period, the toxicity tests are terminated and the LC 3.6.1 Calculations. The LC 3.6.2 Reporting. The test product
and oil and their source and storage are described in the toxicity test report.
Note any observed changes in the experimental water or the test solutions. Also
include the species of fish used; the sources, size, and condition of the fish;
data of any known treatment of the fish for disease or infestation with
parasites before their use; and any observations on the fish behavior at regular
intervals during the tests. In addition to the calculated LC 3.7 Summary of procedures.
3.7.1 Menidia:
1. Prepare adequate stocks of the appropriate standard dilution water.
2. Add 2 liters of the standard dilution water to the test jars. Each test
consists of 5 replicates of each of 5 concentrations of the test material, a
control series of 5 beakers, and a standard reference series of 5 different
concentrations for a total of 35 beakers. Simultaneous performance of toxicity
tests on the oil, product, and oil/product mixture requires a total of 105
beakers.
3. Add the determined amount (quarter points on the log scale) of test
material to the appropriate jars. Preliminary tests will be necessary to define
the range of definitive test concentrations.
4. Cap the jars tightly with the Teflon-lined screw caps and shake for 5
minutes at 315 to 333 2-cm (0.75-inch) strokes per minute on a reciprocal
shaker.
5. Remove the jars from the shaker, take water quality data, dispense 1 liter
of solution to the 1-liter glass beaker, and add 10 acclimated fish per beaker.
6. Aerate with 100±15 bubbles per minute through a 1-ml serological pipette,
as needed, to maintain DO above 4.0 mg/l.
7. Observe and record mortalities, water quality, and behavioral changes
every 24 hours.
8. After 96 hours, terminate the test, and calculate LC 3.7.2 Mysidopsis:
1. Initiate the procedure for hatching the Mysidopsis in sufficient time
before the toxicity test is to be conducted so that 5-7 day old larvae are
available.
2. With the use of a small pipette, transfer 10 Mysidopsis into small
beakers, each containing 200 ml of the proper synthetic seawater.
3. To prepare the test stock product and oil solutions, add 550 ml of the
artificial seawater to the prescribed blender jar. By means of a gas-tight glass
syringe with a Teflon-tipped plunger, add 0.55 ml of the product (or oil) and
mix at 10,000 rpm for 5 seconds. To prepare the test stock oil/product mixture,
add 550 ml of the standard seawater to the blender jar. While the blender is in
operation (10,000 rpm), add 0.5 ml of the oil, then 0.05 ml of the product with
the use of a calibrated syringe with a Teflon-tipped plunger. Blend for 5
seconds after adding the product. One ml of these stock solutions added to the
100 ml of standard seawater in the test containers yields a concentration of 10
ppm product, oil, or oil/product combination (the test will be in a ratio of 1
part product to 10 parts of oil).
4. Each test consists of 5 replications of each of 5 concentrations of the
material under study, a control series of 5 beakers and a standard reference
series of 5 different concentrations, for a total of 35 beakers. Simultaneous
performance of toxicity tests on the oil, product, and oil/product mixture
requires a total of 105 beakers. Immediately after preparing the test solution
of the product or oil/product solution, and using an appropriately sized
syringe, draw up the necessary amount of test solution and dispense into each of
the five containers in each series. Each time a syringe is to be filled for
dispensing to the series of test containers, start the mixer and withdraw the
desired amount in the appropriate syringe while the mixer is in operation. Turn
mixer off immediately after the sample is taken to limit the loss of volatiles.
After adding the required amount of the test oil/product or product mixture,
bring the volume of liquid in each of the test containers up to 800 ml with the
artificial seawater. When the desired concentrations have been prepared, gently
release into each beaker the 10 mysids previously transferred into 200 ml of
medium. This provides a volume of 1 liter in each test chamber.
5. Wash the blender as prescribed for each series of tests.
6. Incubate the test beakers at 25±1 °C for 48 hours with the prescribed
lighting.
7. Terminate the experiment after 48 hours, observe and record the
mortalities, and determine the LC
4.1 Summary of method. The
bioremediation agent effectiveness testing protocol is designed to determine a
product's ability to biodegrade oil by quantifying changes in the oil
composition resulting from biodegradation. The protocol tests for microbial
activity and quantifies the disappearance of saturated hydrocarbons and
polynuclear aromatic hydrocarbons (PAHs). The sample preparation procedure
extracts the oil phase into dichloromethane (DCM), with a subsequent solvent
exchange into hexane. To effectively accomplish the goals of the testing
protocol, it is necessary to normalize the concentration of the various analytes
in oil to a non-biodegradable marker, either C 1Although any of these biomarkers can be used to conduct this
test, it is recommended that hopane be used. 4.2 Apparatus. The following
materials and equipment are required for the protocol: Appropriate flasks and
other glassware; sterile tubes; graduated cylinders (100-ml); deionized water;
p-iodonitrotetrazolium violet dye; weighing pans or paper; 250-ml borosilicate
glass Erlenmeyer flasks with screw tops; Pasteur pipettes; laboratory notebook;
microtiter MPN plates (24-well) multi-channel pipetting device; dilution tube
and caps; autoclave; environmental room or incubator; balance accurate to 0.1 mg
(XD-400); GC/MS instrument equipped with a DB-5 capillary column (30 m, 0.25-mm
I.D., and 0.25-μ m film thickness) and a split/splitless injection port
operating in the splitless mode, such as Hewlett-Packard 5890/5971 GC/MS
(recommended for use); and an autosampler for testing multiple samples.
4.3 Reagents and culture medium.
4.3.1 Preparation of seawater. All
products are tested in clean natural seawater. Clean natural seawater means that
the source of this seawater must not be heavily contaminated with industrial or
other types of effluent. For example, seawater should not be obtained from a
source near shipping channels or discharges of industrial or municipal
wastewater, or with high turbidity. The seawater is used within seven days of
collection. No microbial inoculum is added.
4.3.2 Preparation of oil. A medium
weight crude oil, Alaska North Slope (ANS), is artificially weathered by heating
to 521 °F to remove the light end hydrocarbons prior to experimental start-up
(ANS 521). The method is described in the Draft International Standard ISO/DIS
8708 "Crude Petroleum Oil -- Determination of Distillation Characteristics Using
15 Theoretical Plates Columns" by the International Organization for
Standardization (8). The ANS521 crude oil can be obtained from the National
Environmental Technology Applications Center's (NETAC) Bioremediation Products
Evaluation Center (BPEC), University of Pittsburgh Applied Research Center, 615
William Pitt Way, Pittsburgh, PA, 15238, (412) 826-5511. The crude oil is heated
to 190 °C (374 °F) under atmospheric pressure. The system is then cooled and
placed under vacuum (or under an atmospheric pressure of 20 mm Hg) for the final
distillation to an atmospheric equivalent boiling point of 272 °C (521 °F).
4.3.3 Preparation of mineral nutrient
solution. If a commercial product is strictly a microbial agent and does not
contain its own nutrients, a mineral nutrient solution will be provided if
requested by the product manufacturer or vendor. If a commercial product
contains its own nutrients, no further nutrients will be added. The nutrient
solution is a modified salt solution and is described below.
4.3.3.1 Nutrient preparation:
1. N&P Salts. The following salts are added to distilled water and made
up to a 1,000-ml volume. Adjust final pH to 7.8. The solution is sterilized by
autoclaving at 121 °C at 15 psig for 20 minutes or by filtering through a
sterile 0.22 μ m membrane filter. Na KNO 2. MgSO 3. CaCl 4. FeCl 5. Trace Element Solution. The following salts are added to distilled water
and made up to a 1,000-ml volume. The solution is sterilized by autoclaving at
121 °C at 15 psig for 20 minutes. MnSO H ZnSO (NH The pH of the nutrient solution is adjusted with a pH meter calibrated at
room temperature (approximately 25 °C) using commercial buffers of pH 4.0, 7.0,
and 10.0 (Fisher Scientific), as appropriate, prior to use. The pH is adjusted
with concentrated HCl or 10 M NaOH, as appropriate.
4.3.3.2 Final concentrations: Ten
(10) ml of solution 1 and 2 ml of solutions 2-5 are added to non-sterile
seawater and made up to a 1,000-ml volume immediately prior to test start-up.
This seawater/mineral nutrient solution is used for all flasks containing
products requiring nutrient supplements and for the flasks containing no
commercial additive. Seawater without the above nutrient solutions is used for
products containing their own source of nutrients.
4.4 Pretest preparation.
4.4.1 Experimental setup.
4.4.1.1 The procedure consists of an
experimental shaker flask setup and the specific set of microbiological and
chemical analyses that are performed on individual product samples. The
following test flasks (labeled with unique identifiers) are prepared and set up
on a gyratory shaker at day 0 to reflect the following treatment design: 4.4.1.2 For each test, a sheet listing the
number of flasks, types of controls, number of replicates, product to be tested,
and other information is prepared. The following steps should be adhered to for
the experimental setup:
1. Borosilicate glass Erlenmeyer flasks (250-ml) are thoroughly cleaned and
autoclaved for 20 minutes at 120 °C at 15 psi, then dried in the drying oven.
2. Flasks are labeled with the appropriate code: product or control, sample
day, and letter indicating replicate.
3. 100 ml of seawater is added to each flask.
4. For nutrient and product treatments that require the addition of
nutrients, seawater containing the nutrient solution is prepared.
5. Pasteur pipettes should be sterilized in advance. Break off the tip to
provide a larger opening prior to sterilization.
6. Pour the approximate amount of oil to be used from the large stock bottle
into a sterile beaker. Keep the beaker covered when oil is not being removed.
7. The labeled flasks containing seawater and other additions, as necessary,
are placed on the balance. The flask is tared. The appropriate amount of oil
(0.5 g) is added drop by drop using a sterile Pasteur pipette with the tip
broken off to provide a wider opening. Care is taken to avoid splashing the oil
or getting it on the sides of flasks. Precautions are taken when handling and
charging the flasks to minimize the likelihood of contamination by exogenous
microbes. This includes using a new sterile pipette for each series of flasks.
8. The weight of the oil is recorded in the laboratory notebook.
9. The product is prepared and added to the appropriate flasks according to
the manufacturer's or vendor's instructions.
10. Flasks are carried upright and carefully placed in the holders on the
shaker table to minimize the amount of oil that might adhere to the side of the
flasks. Flasks in which a significant amount of oil is splashed on the sides are
redone.
11. The prepared flasks are shaken at 200 rpm at 20 °C until such time that
they will be removed for sampling.
4.4.2 Sampling. The control and
treatments (nutrient and product flasks) are sampled three times over a 28-day
period: day 0, day 7, and day 28. The entire flask is sacrificed for analysis; a
0.5-ml aliquot is removed from each flask for the microbiological analysis and
the remainder of each flask is used for the chemical analysis. Specific
procedures for both the microbiological and chemical analysis are described
below. At the time of each sampling event, physical observations of each flask
should be recorded.
4.5 Microbiological analysis. To
monitor the viability of the microbial cultures being studied, microbial
enumerations of hydrocarbon degraders are performed at each sampling event using
a microtiter MPN determination. This is used as an indicator of the relative
change in biomass. This test design relies on using growth response as an
indication of enhanced activity as compared to a "no addition" control.
4.5.1 Media preparation. Media for
microbial enumerations are carefully prepared according to manufacturer's or
other instructions and sterilized using appropriate methods.
4.5.1.1 General media treatment:
Buy Bushnell-Haas (B-H) broth in quantities to last no longer than one year. Use
media on a first-in, first-out basis. When practical, buy media in quarter-pound
multiples, rather than one-pound multiples to keep supply sealed as long as
possible. Keep an inventory of media, including kind, amount, lot number,
expiration date, date received, and date opened. Check inventory before
reordering media. Discard media that are caked, discolored, or show other
deterioration.
4.5.1.2 Sterile saline (pH
adjusted):
1. Weigh 30 g of NaCl.
2. Dissolve in enough water to make 1,000 ml.
3. Adjust pH to 8.0 with NaOH (10M and 0.5M).
4. Sterilize by autoclaving for 15 minutes at 15 psig.
4.5.1.3 Standard nutrient concentrate
(add 1 ml to each 100 ml of Bushnell-Haas medium for MPNs):
1. Weigh compounds listed below, dissolve in DIH Potassium Phosphate, monobasic KH Potassium Phosphate, dibasic K Sodium Phosphate, dibasic Na Ammonium Chloride NH Magnesium Sulfate, heptahydrate MgSO Calcium Chloride, dihydrate CaCl Ferric Chloride, hexahydrate FeCl Trace Elements Manganese Sulfate, monohydrate MnSO Boric Acid H Zinc Sulfate, heptahydrate ZnSO Ammonium Moybdate, tetrahydrate (NH 2. Adjust pH to 6.0.
3. Stir solution for approximately 3 hours, then filter through a Buchner
funnel using #1 paper, which will retain approximately 3.8 g of insolubles.
4. Then filter through a 0.45 micron filter into sterile bottles.
5. Cap bottles, label, and store in refrigerator until used.
4.5.1.4 Quality assurance/Quality
control (QA/QC):
1. Periodically check the effectiveness of sterilization using commercially
available tapes or Bacillus stearothermophilus spore suspensions,
following the instructions with these products.
2. Maintain a media log book that includes the dates, kinds and amounts of
media made, pH, and any problems or observations.
3. Before use, check plates and tubes for signs of contamination, drying, or
other problems.
4.5.1.5 Safety/Special precautions:
1. Note any safety or other precautions for particular media.
2. Note precautions to be followed when using the autoclave.
3. Use gloves and other protective clothes when handling media.
4. Use care in handling hot media.
4.5.2 Microbial enumeration.
Standardized techniques for performing Most Probable Number microbial
enumerations are described below.
4.5.2.1 Dilutions:
1. Prior to sacrificing each flask, remove 0.5 ml of water from each flask
and add it to a tube of 4.5 ml sterile phosphate buffer (1:10 dilution) as
prepared in the Standard Methods for the Examination of Water and
Wastewater (9). Using sterile technique, mix and perform serial dilutions
(0.5 ml of previous dilution to 4.5 ml of sterile phosphate buffer) to
10−9 dilution.
4.5.2.2 Inoculating MPN plates (oil
degrader):
1. Prepare sufficient sterile 0.4 M NaCl (23.4 g NaCl/1,000 ml B-H) and B-H
at pH 7.0 to fill the number of wells required for the test (1.75 ml/well).
2. Using sterile technique, add 1.75 ml of B-H broth to each well.
3. Label the top of the plate with the proper dilution for each row.
4. Add 0.1 ml of fluid from each dilution tube to each well in the
appropriate row, starting with the most dilute.
5. After adding the fluid to all the wells, add 20 μ l of sterilized No. 2
fuel oil to the top of each well.
6. Incubate each plate at 20 °C.
7. After 14 days of incubation, add 100 μ l of p-iodotetrazolium violet dye
(50 mg/10 ml of D.I. water) to each well to determine growth.
8. View plates against a white background to determine if color is present.
Development of a purple or pink color upon standing for 45 minutes constitutes a
positive test.
9. Record the number of positive wells and the dilutions at which they occur.
10. Enter data into a computerized enumeration method using "MPN Calculator"
software program (version 2.3 or higher) by Albert J. Klee, U.S. EPA Office of
Research and Development, Risk Reduction Engineering Laboratory, Cincinnati, OH.
4.5.2.3 Quality assurance/Quality
control:
1. Check pH of medium before preparing wells (pH should be approximately
8.0). Adjust pH, if necessary, with dilute NaOH.
2. Keep prepared tetrazolium violet dye solution in the refrigerator in an
amber bottle when not in use.
3. Have all laboratory personnel periodically run MPNs on the same sample to
test precision.
4.5.2.4 Safety/Special precautions:
1. Use sterile technique in preparing solutions, dilutions, plates, and MPN
wells.
2. Do not pipette potentially hazardous solutions by mouth.
3. Autoclave all plates and wells before discarding.
4.6 Chemical analysis of oil
composition.
4.6.1 Sample procedure. After 0, 7,
and 28 days of incubation on a rotary shaker, the appropriate flasks are
sacrificed and extracted with dichloromethane and spiked with a surrogate
recovery standard. A 10-ml aliquot of the DCM layer is used for the gravimetric
analysis. If significant biodegradation is evident in the results of the
gravimetric analysis, then a solvent exchange into hexane takes place prior to
the GC/MS analysis. Follow steps 1-19 below when preparing for the chemical
analysis.
1. After 0, 7, and 28 days of rotary shaking and incubating at 20 °C, the
reaction vessels are sacrificed. Prior to the chemical analysis, a 0.5-ml sample
of the aqueous phase is removed for the microbiological analysis (see Microbial
Enumeration above).
2. A surrogate recovery standard is prepared in the following manner: 1,000
mg of d 3. A 100-μ l aliquot of the surrogate solution is added to each test flask.
The final concentration of surrogates in each flask is approximately 4 ng/μ l of
solvent in the final extract. The aliphatics and marker data should be corrected
for percent recovery of the 5α-androstane surrogate and the aromatics for the
d 4. The contents of the flask are placed into a 250-ml separatory funnel.
5. Measure a total volume of 50 ml DCM for use in the extraction. Use 3 10-ml
fractions to rinse the flask into the funnel and transfer the remaining aliquot
of DCM to the funnel.
6. Stopper and mix vigorously by shaking (approximately 50 times) while
ventilating properly.
7. Each funnel is set aside to allow the DCM and water layers to partition.
This may take 5-10 minutes for some products, or up to 3 hours if the product
has caused the formation of an emulsion.
8. Drain the first 10 ml of the DCM (bottom) layer, collect, cap, uniquely
label, and use for gravimetric analysis (see below). Drain the remaining 40 ml
and dry it by passing it through a funnel packed with anhydrous sodium sulfate.
9. Assemble a Kuderna-Danish (KD) concentrator by attaching a Snyder column
to an evaporation flask with a graduated concentrator tube. Align vertically and
partially immerse concentrator tube in a water bath (10). Set the water bath to
the appropriate temperature to maintain proper distillation.
10. Collect the de-watered extract into the KD concentrator.
11. Evaporate DCM to approximately 10 ml, then add approximately 50 ml of the
exchange solvent (hexane) and concentrate the volume to 10 ml.
12. Rinse the flask into the concentrator tube with 50 ml hexane and
concentrate to 10 ml. Repeat one more time with 50 ml of hexane.
13. Remove concentrator tube with the recovered 10 ml of sample volume. The
heavier residual material should be present as a precipitate (bottom layer).
14. Centrifuge to aid the separation of the hexane from the precipitant
fraction.
15. Place hexane-soluble fraction (top layer) -- approximately 1.0 ml -- into
a GC/MS vial for analysis (see GC/MS Analysis Procedure below). If column
fouling and deterioration of separation characteristics occur, an alumina column
sample cleanup method can be considered (see Alternative GC/MS Sample Cleanup
Procedure below).
16. Analyze by GC/MS using the conditions determined by the U.S. EPA Risk
Reduction Engineering Laboratory, Water and Hazardous Waste Treatment Research
Division, in Cincinnati, OH, which follows U.S. EPA Method 8270 (see GC/MS
Analysis Procedure below).
17. Calculate surrogate recovery. If surrogate recovery is less than 85
percent for the marker relative to the surrogate recovery standard (d 18. Drain the seawater into a storage sample vial/container.
19. Seal the vial with a Teflon-lined cap and store frozen. This water layer
is kept in case additional extractions are necessary.
4.6.2 Gravimetric analysis. The initial means to evaluate the
effectiveness of a bioremediation agent for oil spill response is through
gravimetric analysis. A statistically significant difference (p < 0.05) in
analytical weight of the oil from the control system as compared to the
analytical weight of the oil treated with a bioremediation agent indicates
biodegradation has successfully occurred. Hence, the disappearance of oil should
be accompanied by significant decreases in total oil residue weight of
extractable materials versus a control. If no significant decrease in oil
residue weight is observed, the need to perform further chemical analysis should
be evaluated. Follow steps 1-3 to conduct the gravimetric analysis.
1. The 10 ml of DCM extract (from Sample Procedure step 8 above) is placed in
a small vial and concentrated to dryness by nitrogen blowdown techniques using a
steady stream of nitrogen (pre-purified gas). If the oil is severely
biodegraded, a larger volume of DCM (>10 ml) may be necessary for the
gravimetric analysis.
2. The residue is weighed 3 times for the gravimetric weight of oil. Record
the weight of the oil.
3. Compare statistically (p < 0.05) the weight of the product treatment
versus the weight of the control from each respective time period. If a
significant decrease is observed in the sampling (flask containing
bioremediation agent) weight, then proceed with the remainder of the sample
procedure.
4.6.3 GC/MS analysis. Often,
analysis of saturated and aromatic hydrocarbons by capillary gas chromatography
of DCM extracts leads to column fouling and deterioration of separation
characteristics. An alternative, simple "one-step" alumina sample cleanup
procedure can be performed on oil before injection; this cleanup removes both
asphaltenes and polar compounds and can be applied to DCM extracts as well. This
procedure is described in steps 1-11 below.
4.6.3.1 Alternative GC/MS sample
cleanup procedure:
1. Weigh 4.0 g alumina (neutral, 80-200 mesh) into scintillation vials
covered loosely with aluminum foil caps. Prepare one scintillation vial per
sample. Heat for 18 hours at 300 °C or longer. Place in a desiccator of silica
until needed.
2. Add 5.0 ml of DCM to a glass luerlok multi-fit syringe (e.g., BD #2471)
with stopcock (e.g., Perfectum #6021) in closed position, stainless steel
syringe needle (18 gauge), and PTFE frits. Clamp in a vertical position.
3. Transfer 4.0 g of prepared alumina to a plastic weighing boat and fill
syringe slowly while applying continuous vibration (e.g., Conair # HM 11FF1).
4. Add a second PTFE frit and push into place on top of the alumina bed.
5. Drain 5.0 ml DCM to the top level of the column frit to await sample
addition and discard DCM.
6. Weigh 50 mg ± 0.1 mg ANS521 oil into a tared vial.
7. Premeasure 10 ml of DCM into a graduated cylinder. Add 0.2 to 0.3 ml of
the DCM to the tared oil vial. Mix and transfer solvent to the column bed with a
Pasteur pipette. Open stopcock and collect in a 10-ml volumetric flask. Repeat
until approximately 1.0 ml (do not exceed 1.0 ml) of DCM has rinsed the vial and
inner walls of the syringe body into the 10-ml flask.
8. Transfer balance of DCM from the graduated cylinder to the column and
regulate the solvent flow rate to approximately 1 to 2 ml/minute. Collect all
eluent in the 10-ml flask.
9. Transfer a known volume of eluent to another scintillation vial and blow
down to dryness (nitrogen).
10. Determine and record weight.
11. Dissolve in 1.0 ml hexane for the GC/MS analysis procedure (see below).
4.6.3.2 GC/MS analysis procedure:
Immediately prior to injection, an internal standard solution of four
deuterated compounds is spiked into the sample extracts and injected. Samples
are quantified using the internal standard technique (10) for both the aliphatic
and aromatic fractions of the oil extracts in order to provide sufficient
information that the oil is being degraded. To help ensure that the observed
decline in target analytes is caused by biodegradation rather than by physical
loss from mishandling or inefficient extraction, it is necessary to normalize
the concentrations of the target analytes via a "conserved internal marker."
Conserved internal markers that have been found useful for quantification are
C 1. One (1) ml of the hexane extract (from Sample Procedure step 15 above) is
placed into a 1.5-ml vial for use on the autosampler of the GC/MS instrument.
2. To this solution, 20 μl of a 500-ng/μl solution of the internal standards
is added and the vial is capped for injection. The final concentration of the
internal standards in each sample is 10 ng/μl. This solution contains 4
deuterated compounds: d 3. At the start of any analysis period, the mass spectrometer (MS) is tuned
to PFTBA by an autotune program, such as the Hewlett-Packard quicktune routine,
to reduce operator variability. Set the GC/MS in the SIM mode at a scan rate of
1.5 scans/second to maximize the linear quantitative range and precision of the
instrument. Set all other conditions to those specified in Instrument
Configuration and Calibration section below.
4. An instrument blank and a daily standard are analyzed prior to analysis of
unknowns. Internal standards are combined with the sample extracts and
coinjected with each analysis to monitor the instrument's performance during
each run.
5. Information that should be included on the acquisition form include
operator's name and signature, date of extraction, date and time of autotune,
date of injection(s), instrument blank, daily standard mix injection, GC column
number, and standards for the 5-point calibration curve.
6. If the instrument is operated for a period of time greater than 12 hours,
the tune will be checked and another daily standard analyzed prior to continuing
with analyses. 7. The MS is calibrated using a modified version of EPA Method 8270 (10).
Specifically, the concentrations of internal standards are 10 ng/μl instead of
40 ng/μl. A five-point calibration curve is obtained for each compound listed in
table 6 prior to sample analysis at 1, 5, 10, 25, and 50 ng/μl. A 5-point
calibration must be conducted on a standard mix of compounds to determine RRFs
for the analytes. The standard mix (excluding the marker) for this calibration
curve may be obtained from Absolute Standards, Inc., 498 Russell St., New Haven,
CT, 06513, (800) 368-1131. If C 8. Calculate each compound's relative response factor to its corresponding
deuterated internal standard indicated above, using the following equation: RRF=(A where: RRF=relative response factor
A A C C 9. Identify each analyte based on the integrated abundance from the primary
characteristic ion indicated in table 7.
10. Quantitate each analyte using the internal standard technique. The
internal standard used shall be the one nearest the retention time of that of a
given analyte (Table 8). 11. Use equation 7 to calculate the concentration of analytes in ng/mg (ppm)
oil: Concentration (ng/mg)=(A where: A I V A RRF=relative response factor
V M 12. Compute the "normalized concentrations" for each target analyte
concentration at a given sampling time (equation 7) by simply dividing by the
conserved internal marker concentration at the same sampling time.
4.6.4 Generally accepted laboratory
procedures. Samples are immediately logged into the laboratory, where they
will be given a unique sample identification based on Julian data and the number
logged in. Prior to the analysis of any experimental samples, a five-point
standard curve is prepared. One of the mid-range standard curve concentration
levels is analyzed daily before sample analysis as a continuing standard. RRFs
for all target analytes should be within 25% of the standard curve
response values at day 0, and at any sampling event the check standard percent
difference from the initial five-point calibration must not exceed 20% between
the before and after daily standard mix (see below). The collected GC/MS data
are initially processed by a macro routine, which performs extracted
chromatographic plots of the target compounds, integrates the target compounds,
and shows integration results to include tabular numbers. The integration values
are then transferred to a spreadsheet format to be quantified. Because of the
complexity of the analyte matrix (oil), a very high degree of manual
verification and reintegration of the spectral data is required.
4.6.5 QA/QC procedures. The
reliability of this method is dependent on the QA/QC procedures followed. Before
and after each analytical batch (approximately 10 samples), analyze one
procedural blank, one duplicate, and one calibration verification standard (10
ng/μ l). Analyze one reference crude oil standard. The instrument's performance
and reproducibility are validated routinely in this manner. Surrogate recoveries
should be within 70 to 120%, and duplicate relative percent difference values
should be ±20%. A control chart of the standard oil should be prepared and
monitored. Variations of analytes in the control chart should be no more than
25% from the historical averages. Injection port discrimination for n-C25 and
greater alkanes must be carefully monitored; the ratio of RRF n-C32/RRF n-C21
alkanes should not be allowed to fall below 80%. The mass discrimination can be
reduced by replacing the quartz liner in the injection port after every
analytical batch. The instrument's performance and reproducibility are validated
routinely by analyzing the reference crude oil standard. All analyses are
recorded in instrument logs detailing operating conditions, date and time, file
name, etc. After analysis, the sample extracts are archived at refrigeration
temperatures. To document QA/QC, the following information is contained in the
detailed quantitative reports: average RRF derived from the standard curve; RRF
from the daily standard; percent relative standard deviation; area of target
analyte; concentration determined both on a weight and volume basis; and values
for any surrogates and internal standards.
4.6.6 Instrument configuration and
calibration. A 2-ml aliquot of the hexane extract prepared by the above
procedure is injected into a GC/MS instrument, such as the Hewlett-Packard
5890/5971 GC/MS (recommended for use). This instrument should be equipped with a
DB-5 capillary column (30 m, 0.25-mm I.D., and 0.25-μ m film thickness) and a
split/splitless injection port operating in the splitless mode. Table 9
summarizes the temperature program used for the analysis. This temperature
program has been optimized to give the best separation and sensitivity for
analysis of the desired compounds on the instrument. Prior to the sample
analysis, a five-point calibration must be conducted on a standard mix of the
compounds listed in table 7 to determine RRFs for the analyses. 4.7 Statistical analysis. The
determination of a bioremediation agent's effectiveness will be partially based
upon the results of a statistical analysis of the shaker flask experiment. The
experimental design for this test is a two factorial design. This two-way
analysis of variance (ANOVA) will be used to determine data trends. The
statistical method is designed to test various types of bioremediation
treatments including microbial, nutrient, enzyme, and combination products. The
following is a summary of the statistical methods to be used to evaluate the
analytical data obtained from all product tests. The experimental design, data
analysis methodology, interpretation of results, required documentation, and a
numeric example are outlined below.
4.7.1 Experimental design. The
experimental design for this test is known as a factorial experiment with two
factors. The first factor is product/control group; the second factor is time
(measured in days). For example, if two groups (product A and a non-nutrient
control) are tested at each of three points in time (day 0, 7, and 28), the
experiment is called a 2x3 factorial experiment. There will be three
replications (replicated shaker flasks) of each group-time combination.
4.7.2 Data analysis methods. For
each analyte and each product used, a product is considered a success by the
demonstration of a statistically significant difference between the mean analyte
degradation by the product and the mean analyte degradation by the non-nutrient
control. Such a determination will be made by performing an ANOVA on the sample
data. The technical aspects of this procedure are outlined in Snedecor and
Cochran (12). Most statistical software packages support the use of two-way
ANOVA. However, the format required for the input data differs among the various
commercial packages. Whichever package is used, the following ANOVA table will
be provided as part of the output. In the Degree of Freedom column of table 10,
p = the number of product/control groups, t = the number of days at which each
group is analyzed, and n = the number of replications. For the example of the
2x3 factorial experiment discussed above, p=2, t=3, and n=3. The significance of
the F-statistics (as indicated by their corresponding p-values) are used to
interpret the analysis. 4.7.3 Interpretation. 4.7.3.1 If
the F-statistic for the interaction is significant at the 0.05 level (i.e.,
p-value is less than 0.05), the data indicate that the mean response of at least
two groups being tested differ for at least one point in time. In order to find
out which groups and at which points in time the difference occurs, pairwise
comparisons between the group means should be conducted for all time points.
These comparisons can be made using protected least squared difference (LSD) or
Dunnett mean separation techniques. The protected LSD procedure is detailed in
Snedecor and Cochran (12); the Dunnett procedure is outlined in Montgomery (13).
For both methods, the mean square error (MSE) from the two-way ANOVA table
should be used to compute the separation values.
4.7.3.2 If the F-statistic for the
interaction is not significant at the 0.05 level (i.e., p-value not less than
0.05), but the F-statistic for the group is significant (i.e., p-value is less
than 0.05), the data indicate that any differences that exist among the group
means are consistent across time. To find out which group means differ, a
pairwise comparison of the group means should be carried out by pooling data
across all points in time. Again, the MSE from the two-way ANOVA table should be
used to compute the separation values.
4.7.3.3 If the F-statistic corresponding
to both interaction and group are not significant at the 0.05 level, the data
indicate no difference between the group means at any point in time. In this
case, no further analysis is necessary.
4.7.3.4 Finally, Snedecor and Cochran (12)
use caution concerning the use of multiple comparisons. If many such comparisons
are being conducted, then about 5% of the tested differences will erroneously be
concluded as significant. The researcher must guard against such differences
causing undue attention.
4.7.4 Required documentation.
4.7.4.1 The following documents should be included to summarize the findings
from a product test.
1. Data listings for each analyte that was analyzed. These should show all
raw data.
2. A table of summary statistics for each analyte. The table should include
the mean, standard deviation, and sample size for each group at each day.
3. An ANOVA table for each analyte. The table should be of the same format as
table 10.
4. A clear summary of the mean separations (if mean separations were
necessary). The mean separation methods (LSD or Dunnett), the significance
level, the minimum significant difference value, and the significant differences
should be clearly marked on each output page.
5. All computer outputs should be included. No programming alterations are
necessary. The specific computer package used to analyze the data should be
included in the report.
Example. An analysis of the total aromatic data (in ppm) was conducted
for the following three groups:
Group 1: Non-nutrient Control
Group 2: Nutrient Control
Group 3: Test Product
4.7.4.2 The raw data are shown in table
11. Note the three replications for each group-time combination. 4.7.4.3 Table 12 gives the summary
statistics (number of observations, means, and standard deviations) for each
group-time combination. 4.7.4.4 Table 13 shows the results of the
two-way ANOVA. 4.7.4.5 From table 13, it can be seen that
the F-statistic for interaction is significant (F=61.39, p=0.0001). This
indicates that group differences exist for one or more days. Protected LSD mean
separations were then conducted for each day to determine which group
differences exist. The results are summarized in table 14. Note that means with
the same letter (T grouping) are not significantly different. 4.7.4.6 The grouping letters indicate that
the product mean values (group 3) at day 7 and day 28 are significantly
different from those of both the nutrient control (group 2) and the non-nutrient
control (group 1) for those days. No other significant differences are shown.
Therefore, in terms of total aromatic degradation, the test indicates the
desired statistically significant difference between the mean of the product and
the mean of the non-nutrient control.
The purpose of this format is to summarize in a standard and convenient
presentation the technical product test data required by the U.S. Environmental
Protection Agency before a product may be added to EPA's NCP Product Schedule,
which may be used in carrying out the National Oil and Hazardous Substances
Pollution Contingency Plan. This format, however, is not to preclude the
submission of all the laboratory data used to develop the data summarized in
this format. Sufficient data should be presented on both the effectiveness and
toxicity tests to enable EPA to evaluate the adequacy of the summarized data. A
summary of the technical product test data should be submitted in the following
format. The numbered headings should be used in all submissions. The subheadings
indicate the kinds of information to be supplied. The listed subheadings,
however, are not exhaustive; additional relevant information should be reported
where necessary. As noted, some subheadings may apply only to particular types
of agents.
I. Name, Brand, or Trademark
II. Name, Address, and Telephone Number of Manufacturer
III. Name, Address, and Telephone Numbers of Primary Distributors
IV. Special Handling and Worker Precautions for Storage and Field
Application
1. Flammability.
2. Ventilation.
3. Skin and eye contact; protective clothing; treatment in case of contact.
4. Maximum and minimum storage temperatures; optimum storage temperature
range; temperatures of phase separations and chemical changes.
V. Shelf Life
VI. Recommended Application Procedure
1. Application method.
2. Concentration, application rate (e.g., gallons of dispersant per ton of
oil).
3. Conditions for use: water salinity, water temperature, types and ages of
pollutants.
VII. Toxicity (Dispersants, Surface Washing Agents, Surface Collecting
Agents, and Miscellaneous Oil Spill Control Agents) VIII.(a). Effectiveness (bioremediation agents). Raw data must be
reported according to the format shown below. The first column lists the names
of the analytes measured by GC/MS (SIM), the surrogate standards, and various
ratios and sums. In the next three columns, the concentration of the analytes
(ng/mg oil), the concentration of the analytes corrected for the recovery of the
surrogate standard (α-androstane for alkanes, d
Table 1_Major Ion Composition of ``Instant Ocean'' Synthetic Sea Salt
------------------------------------------------------------------------
Ionic
Concentration
Major Ion % Total at 34 ppt
Weight salinity (mg/
1)
------------------------------------------------------------------------
Chloride (C1[SU]-[/SU])...................... 47.470 18,740
Sodium (NA[SU]=[/SU])........................ 26.280 10,454
Sulfate (SO[INF]4[/INF]-).................... 6.602 2,631
Magnesium (Mg[SU]==[/SU]).................... 3.230 1,256
Calcium (Ca[SU]==[/SU])...................... 1.013 400
Potassium (K[SU]=[/SU])...................... 1.015 401
Bicarbonate (HCO[INF]3[/INF]-)............... 0.491 194
Boron (B).................................... 0.015 6.0
Strontium (Sr[SU]==[/SU]).................... 0.001 7.5
SOLIDS TOTAL................................ 86.11% 34,089.50
Water........................................ 13.88
TOTAL....................................... 99.99%
------------------------------------------------------------------------
Table 2_Test Oil Characteristics
------------------------------------------------------------------------
Prudhoe Bay crude South Louisiana
oil crude oil
------------------------------------------------------------------------
Specific gravity \1\........... 0.894 kg/1......... 0.840 kg/1
API gravity \1\................ 26.8 degrees....... 37.0 degrees
Sulfur......................... 1.03 wt%........... 0.23 wt%
Sulfur compounds, profile...... ................... ..................
Nitrogen....................... 0.20 wt%........... 0.031 wt%
Vanadium....................... 21 mg/1............ 0.95 mg/1
Nickel......................... 11 mg/1............ 1.1 mg/1
Simulated distillation profile. ................... ..................
Infrared spectrum.............. ................... ..................
UV fluorescence spectrum....... ................... ..................
Pour Point..................... +25 °F........ 0 °F
Viscosity
at 40 °C................. 14.09 cST.......... 3.582 cST
at 100 °C................ 4.059 cST.......... 1.568 cST
Index.......................... 210................ ([SU]2[/SU])
------------------------------------------------------------------------
\1\ At 15 °C
\2\ Not calculable when viscosity at 100 °C is less than 2.0.
Table 3_Oil Standard Solutions: Concentrations in Final DCM Extractions
\1\
------------------------------------------------------------------------
Volume of
Final oil Final extract Total amount of parent oil-DCM
concentration (mg/ml volume (ml of oil in standard std (ml) added
of DCM) DCM) (mg) to saltwater
------------------------------------------------------------------------
4.0 20.0 80.0 890
2.0 20.0 40.0 440
1.0 20.0 20.0 220
0.50 20.0 10.0 110
0.10 20.0 2.0 22
0.05 20.0 1.0 11
------------------------------------------------------------------------
\1\ Assuming an oil density of 0.9 g/ml and an extraction efficiency of
100% for oil from the 30-ml of seawater.
Table 4_Synthetic Seawater
[Toxicity Test]
------------------------------------------------------------------------
Salt (g) \1\
------------------------------------------------------------------------
NaF........................................................ 1.9
SrCl[INF]2[/INF] · 6H[INF]2[/INF]O.................... 13.0
H[INF]3[/INF] BO[INF]2[/INF]............................... 20.0
KBr........................................................ 67.0
KCl........................................................ 466.0
CaC1[INF]2[/INF] · 2H[INF]2[/INF]O.................... 733.0
Na[INF]2[/INF] SO[INF]4[/INF].............................. 2,660.0
MgCl[INF]2[/INF] · 6H[INF]2[/INF]O.................... 3,330.0
NaCl....................................................... 15,650.0
Na[INF]2[/INF]SiO[INF]3[/INF] · 9H[INF]2[/INF]O....... 13.0
EDTA[SU]2[/SU]............................................. 0.4
NaHCO[INF]3[/INF].......................................... 133.0
------------------------------------------------------------------------
\1\ Amount added to 900 liters of water, as described in the text.
\2\ Ethylenediaminetetraacetate tetrasodium salt.
Table 5_Test Oil Characteristics: No. 2 Fuel Oil
------------------------------------------------------------------------
Characteristic Minimum Maximum
------------------------------------------------------------------------
Gravity (°API)................................. 32.1 42.8
Viscosity kinematic at 100 °F (cs)............. 2.35 3.00
Flash point ( °F).............................. 150 ...
Pour point ( °F)............................... ... 0
Cloud point ( °F).............................. ... 10
Sulfur (wt %)....................................... ... 0.35
Aniline point ( °F)............................ 125 180
Carbon residue (wt %)............................... ... 0.16
Water (vol %)....................................... ... 0
Sediment (wt %)..................................... ... 0
Aromatics (vol %)................................... 10 15
Distillation:
IBP ( °F)..................................... 347 407
10% ( °F)..................................... 402 456
50% ( °F)..................................... 475 530
90% ( °F)..................................... 542 606
End Point ( °F)............................... 596 655
Neutralization No................................... ... 0.05
------------------------------------------------------------------------
----------------------------------------------------------------------------------------------------------------
No. of samples at sampling Total No. of analytical
times determinations
Treatment -------------------------------------------------------------------
Microbial
Day 0 Day 7 Day 28 counts Gravimetric GC/MS
----------------------------------------------------------------------------------------------------------------
Control..................................... 3 3 3 9 9 9
Nutrient.................................... 3 3 3 9 9 9
Product..................................... 3 3 3 9 9 9
----------------------------------------------------------------------------------------------------------------
Control = Oil + Seawater
Nutrient = Oil + Seawater + Nutrient
Product = Oil + Seawater + Product (+ Nutrient, if required).
Table 6_Analytes Listed Under the Corresponding Internal Standard Used for Calculating RRFs
----------------------------------------------------------------------------------------------------------------
d[INF]8[/INF]- d[INF]10[/INF]- d[INF]12[/INF]- d[INF]12[/INF]-
Internal Standard naphthalene anthracene chrysene perylene
----------------------------------------------------------------------------------------------------------------
Alkanes......................... nC10-nC15........... nC16-nC23........ nC24-nC29........ nC30-nC35.
Pristane......... ................. C[INF]30[/
INF]17[beta](H),
21[alpha](H)-
hopane.
Phytane.......... ................. ..................
5[alpha]- ................. ..................
androstane.
Aromatics....................... Naphthalene......... Dibenzothiophene. Fluoranthene..... Benzo(b)fluoranthe
ne.
Fluorene......... Pyrene........... Benzo(k)fluoranthe
ne.
Anthracene....... Chrysene......... Benzo(e)pyrene.
Phenanthrene..... ................. Benzo(a)pyrene.
Perylene.
Indeno(g,h,i)pyren
e.
Dibenzo(a,h)
anthracene.
Benzo(1,2,3-
cd)perylene.
----------------------------------------------------------------------------------------------------------------
Table 7_Primary Ions Monitored for Each Target Analyte During GC/MS
Analysis
------------------------------------------------------------------------
Compound Ion
------------------------------------------------------------------------
n-alkanes (C[INF]10[/INF]-C[INF]35[/INF]).......................... 85
Pristane........................................................... 85
Phytane............................................................ 85
Naphthalene........................................................ 128
C1-naphthalenes.................................................... 142
C2-naphthalenes.................................................... 156
C3-naphthalenes.................................................... 170
C4-naphthalenes.................................................... 184
Fluorene........................................................... 166
C1-fluorenes....................................................... 180
C2-fluorenes....................................................... 194
C3-fluorenes....................................................... 208
Dibenzothiophenes.................................................. 184
C1-dibenzothiophenes............................................... 198
C2-dibenzothiophenes............................................... 212
C3-dibenzothiophenes............................................... 226
Anthracene......................................................... 178
Phenanthrene....................................................... 178
C1-phenanthrenes................................................... 192
C2-phenanthrenes................................................... 206
C3-phenanthrenes................................................... 220
Fluoranthene/pyrene................................................ 202
C1-pyrenes......................................................... 216
C2-pyrenes......................................................... 230
Chrysene........................................................... 228
C1-chrysenes....................................................... 242
C2-chrysenes....................................................... 256
Hopanes (177 family)............................................... 177
Hopanes (191 family)............................................... 191
Steranes (217 family).............................................. 217
Benzo(b)fluoranthene............................................... 252
Benzo(k)fluoranthene............................................... 252
Benzo(e)pyrene..................................................... 252
Benzo(a)pyrene..................................................... 252
Perylene........................................................... 252
Ideno(g,h,i)pyrene................................................. 276
Dibenzo(a,h)anthracene............................................. 278
Benzo(1,2,3-cd)perylene............................................ 276
d[INF]8[/INF]-naphthalene.......................................... 136
d[INF]10[/INF]-anthracene.......................................... 188
d[INF]10[/INF]-phenanthrene........................................ 188
d[INF]12[/INF]-chrysene............................................ 240
d[INF]12[/INF]-perylene............................................ 264
[alpha]-androstane................................................. 260
------------------------------------------------------------------------
Table 8_Analytes and Reference Compounds
----------------------------------------------------------------------------------------------------------------
Compound Reference compound Compound Reference compound
----------------------------------------------------------------------------------------------------------------
n-C10................................ n-C10.................. C2-naphthalene......... Naphthalene.
n-C11................................ n-C11.................. C3-naphthalene......... Naphthalene.
n-C12................................ n-C12.................. C4-naphthalene......... Naphthalene.
n-C13................................ n-C13.................. Fluorene............... Fluorene.
n-C14................................ n-C14.................. C1-fluorene............ Fluorene.
n-C15................................ n-C15.................. C2-fluorene............ Fluorene.
n-C16................................ n-C16.................. C3-fluorene............ Fluorene.
n-C17................................ n-C17.................. Dibenzothiophene....... Dibenzothiophene.
Pristane............................. Pristane............... C1-dibenzothiophene.... Dibenzothiophene.
n-C18................................ n-C18.................. C2-dibenzothiophene.... Dibenzothiophene.
Phytane.............................. Phytane................ C3-dibenzothiophene.... Dibenzothiophene.
n-C19................................ n-C19.................. Phenanthrene........... Phenanthrene.
n-C20................................ n-C20.................. Anthracene............. Anthracene.
n-C21................................ n-C21.................. C1-phenanthrene........ Phenanthrene.
n-C22................................ n-C22.................. C2-phenanthrene........ Phenanthrene.
n-C23................................ n-C23.................. C3-phenanthrene........ Phenanthrene.
n-C24................................ n-C24.................. Fluoranthene........... Fluoranthene.
n-C25................................ n-C25.................. Pyrene................. Pyrene.
n-C26................................ n-C26.................. C1-pyrene.............. Pyrene.
n-C27................................ n-C27.................. C2-pyrene.............. Pyrene.
n-C28................................ n-C28.................. Chrysene............... Chrysene.
n-C29................................ n-C29.................. C1-chrysene............ Chrysene.
n-C30................................ n-C30.................. C2-chrysene............ Chrysene.
n-C31................................ n-C31.................. Benzo(b)fluoranthene... Benzo(b)fluoranthene.
n-C32................................ n-C32.................. Benzo(k)fluoranthene... Benzo(k)fluoranthene.
n-C33................................ n-C33.................. Benzo(e)pyrene......... Benzo(e)pyrene.
n-C34................................ n-C34.................. Benzo(a)pyrene......... Benzo(a)pyrene.
n-C35 C[INF]30[/ n-C35 C[INF]30[/ Perylene Perylene
INF]17[alpha],21[beta]-hopane. INF]17[alpha],21[beta]- ideno(g,h,i)pyrene. ideno(g,h,i)pyrene.
hopane.
5[alpha]-androstane.................. 5[alpha]-androstane.... Dibenzo(a,h)anthracene. Dibenzo(a,h)anthracene.
C1-naphthalene....................... Naphthalene............ Benzo(1,2,3-cd)perylene Benzo(1,2,3-
cd)perylene.
----------------------------------------------------------------------------------------------------------------
Table 9_Operating Conditions and Temperature Program of GC/MS
------------------------------------------------------------------------
Operating conditions
-------------------------------------------------------------------------
Injector port_290 °C
Transfer line_320 °C
Total run time_73 minutes
Column flow rate (He)_1.0 ml/minute
------------------------------------------------------------------------
Temperature Program
-----------------------------------------------------------------------------------------------------------------
Temp. Rate,
Level 1, Time 1, °C/ Temp 2, Time 2,
°C minutes minute °C minutes
----------------------------------------------------------------------------------------------------------------
Level 1........................................................... 55 3 5 280 5
Level 2........................................................... 280 0 3 310 10
----------------------------------------------------------------------------------------------------------------
Table 10_Two-Way ANOVA Table
----------------------------------------------------------------------------------------------------------------
Degree of freedom
Source (df) Sum of squares Mean square F-Statistic p-Value
----------------------------------------------------------------------------------------------------------------
Group........................ p-1 SSG MSG-MSG/MSE MSG/MSE \1\
Time......................... t-1 SST MST-MST/MSE MST/MSE \1\
Interaction.................. (p-1)(t-1) SSI MSI-MSI/MSE MSI/MSE \1\
Error........................ pt(n-1) SSE MSE-SSE
Total.................. npt-1 SSTOT
----------------------------------------------------------------------------------------------------------------
\1\ To be determined from the value of the F-statistic.
Table 11_Product Test Data, Total Aromatics (ppm)
------------------------------------------------------------------------
Group 1 Group 2 Group 3
------------------------------------------------------------------------
Day 0........................................ 8153 7912 7711
8299 8309 8311
8088 8111 8200
Day 7........................................ 8100 7950 6900
8078 8200 6702
7999 8019 5987
Day 28....................................... 8259 8102 4000
8111 7754 3875
8344 7659 3100
------------------------------------------------------------------------
Table 12_Summary Statistics for Product Test Data Total Aromatics (ppm)
------------------------------------------------------------------------
Standard
Time Product n Mean deviation
------------------------------------------------------------------------
Day 0................................ Group 1 3 8,180.0 108.1
Group 2 3 8,110.7 198.5
Group 3 3 8,074.0 319.2
Day 7................................ Group 1 3 8,059.0 53.1
Group 2 3 8,056.3 129.1
Group 3 3 6,529.7 480.3
Day 28............................... Group 1 3 8,238.0 117.9
Group 2 3 7,838.3 233.2
Group 3 3 3,658.3 487.6
------------------------------------------------------------------------
Table 13_Example Two-Way ANOVA Table
----------------------------------------------------------------------------------------------------------------
F-
Source df Sum of squares Mean square statistic p-value
----------------------------------------------------------------------------------------------------------------
Group.............................................. 2 23,944,856.41 11,972,428.70 151.94 0.0001
Time............................................... 2 10,954,731.19 5,477,365.59 69.51 0.0001
Interaction........................................ 4 19,347,589.04 4,836,897.26 61.39 0.0001
Error.............................................. 18 1,418,303.33 78,794.63 ......... .......
------------------------------------------------------------
Total........................................ 26 55,665,480.96 ............... ......... .......
----------------------------------------------------------------------------------------------------------------
Table 14_Pairwise Protected LSD Mean Separation
------------------------------------------------------------------------
T grouping Mean n Interaction
------------------------------------------------------------------------
A.............................. 8,338.0 3 Group 1, Day 28.
A.............................. 8,180.0 3 Group 1, Day 0.
A.............................. 8,110.7 3 Group 2, Day 0.
A.............................. 8,074.0 3 Group 3, Day 0.
A.............................. 8,059.0 3 Group 1, Day 7.
A.............................. 8,056.3 3 Group 2, Day 7.
A.............................. 7,838.3 3 Group 2, Day 28.
B.............................. 6,529.7 3 Group 3, Day 7.
C.............................. 3,658.3 3 Group 3, Day 28.
------------------------------------------------------------------------
Significant Level = 0.05.
Degrees of Freedom = 18.
Mean Square Error = 78794.63.
Critical Value = 2.10.
Least Significant Difference = 481.52.
------------------------------------------------------------------------
Materials Tested Species LC[INF]50[/INF] (ppm)
------------------------------------------------------------------------
Product Menidia beryllina 96-hr.
Mysidopsis bahia 2 48-hr.
No. 2 fuel oil Menidia beryllina 96-hr.
Mysidopsis bahia 48-hr.
Product and No. 2 Menidia beryllina 96-hr.
fuel oil (1:10) Mysidopsis bahia 48-hr.
------------------------------------------------------------------------
Bioremediation Agent Effectiveness Test Raw Data
[Date: . Testing Date: 0, 7, 28 (Circle One). Initial Oil Weight: .]
----------------------------------------------------------------------------------------------------------------
Product Replicate 1
--------------------------------------------------------- Product Replicate
Concentration ng/ Surrogate Normalized to 2
mg corrected ng/mg marker ng/mg
----------------------------------------------------------------------------------------------------------------
Alkane Analyte ................. ................. ................. .................
n-C10........................... ................. ................. ................. .................
n-C11........................... ................. ................. ................. .................
n-C12........................... ................. ................. ................. .................
n-C13........................... ................. ................. ................. .................
n-C14........................... ................. ................. ................. .................
n-C15........................... ................. ................. ................. .................
n-C16........................... ................. ................. ................. .................
n-C17........................... ................. ................. ................. .................
pristane........................ ................. ................. ................. .................
n-C18........................... ................. ................. ................. .................
phytane......................... ................. ................. ................. .................
n-C19........................... ................. ................. ................. .................
n-C20........................... ................. ................. ................. .................
n-C21........................... ................. ................. ................. .................
n-C22........................... ................. ................. ................. .................
n-C23........................... ................. ................. ................. .................
n-C24........................... ................. ................. ................. .................
n-C25........................... ................. ................. ................. .................
n-C26........................... ................. ................. ................. .................
n-C27........................... ................. ................. ................. .................
n-C28........................... ................. ................. ................. .................
n-C29........................... ................. ................. ................. .................
n-C30........................... ................. ................. ................. .................
n-C31........................... ................. ................. ................. .................
n-C32........................... ................. ................. ................. .................
n-C33........................... ................. ................. ................. .................
n-C34........................... ................. ................. ................. .................
n-C35........................... ................. ................. ................. .................
n-C36........................... ................. ................. ................. .................
[alpha]-androstane.............. ................. ................. ................. .................
Total alkanes................... ................. ................. ................. .................
n-C17:pristane.................. ................. ................. ................. .................
n-C18:phytane................... ................. ................. ................. .................
Aromatic Analyte: ................. ................. ................. .................
naphthalene..................... ................. ................. ................. .................
C1-naphthalenes................. ................. ................. ................. .................
C2-naphthalenes................. ................. ................. ................. .................
C3-naphthalenes................. ................. ................. ................. .................
C4-naphthalenes................. ................. ................. ................. .................
dibenzothiophene................ ................. ................. ................. .................
fluorene........................ ................. ................. ................. .................
C1-fluorenes.................... ................. ................. ................. .................
C2-fluorenes.................... ................. ................. ................. .................
C3-fluorenes.................... ................. ................. ................. .................
C1-dibenzothiophenes............ ................. ................. ................. .................
C2-dibenzothiophenes............ ................. ................. ................. .................
C3-dibenzothiophenes............ ................. ................. ................. .................
phenanthrene.................... ................. ................. ................. .................
anthracene...................... ................. ................. ................. .................
C1-phenanthrenes................ ................. ................. ................. .................
C2-phenanthrenes................ ................. ................. ................. .................
C3-phenanthrenes................ ................. ................. ................. .................
naphthobenzothio................ ................. ................. ................. .................
C1-naphthobenzothio............. ................. ................. ................. .................
C2-naphthobenzothio............. ................. ................. ................. .................
C3-naphthobenzothio............. ................. ................. ................. .................
fluoranthene.................... ................. ................. ................. .................
pyrene.......................... ................. ................. ................. .................
C1-pyrenes...................... ................. ................. ................. .................
C1-pyrenes...................... ................. ................. ................. .................
chrysene........................ ................. ................. ................. .................
benzo(a)anthracene.............. ................. ................. ................. .................
C1-chrysenes.................... ................. ................. ................. .................
c2-chrysenes.................... ................. ................. ................. ......
