EPA R2-73-201
MAY 1973 Environmental Protection TecSinalogy Series
Standard Dispersant Effectiveness
and Toxicity Tests
National Environmental Research Center
Office af Research and Monitoring
U.S. Envsronmenta! Protection
Cincinnati, Ohio 45263
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RESIAECH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
degradation from point and non-point sources of
pollution. This work provides the new or improved
technology required for the control and treatment
of pollution sources to meet environmental quality
standards.
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EPA-R2-73-201
May 1973
STANDARD DISPERSANT EFFECTIVENESS
AND TOXICITY TESTS.
L. T. McCarthy, Jr., I. Wilder and
J. S. Dorrler
Edison Water Quality Research Laboratory
Edison, New Jersey 08817
Program Element 1B2041
National Environmental Research Center
Office of Research and Monitoring
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
For sale by the Superintendent or Documents, U.S. Government Printing Office, Washington, D.C. 20402
Price 90 cents domestic postpaid or 65 cents QPO Bookstore
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REVIEW NOTICE
The National Environmental Research Center, Cincinnati, has
reviewed this report and approved its publication. Mention
of trade names or commercial products does not constitute
endorsement or recommendation for use.
ii
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ABSTRACT
A brief history of the development of the Standard EPA Dispersant Effec-
tiveness and Toxicity tests is outlined. The standard tests are pre-
sented and discussed. An analysis of variance is performed on the data
developed by three independent laboratories in order to determine the
reproducibility of standard test procedures.
In the standard effectiveness test, oil is applied to the water surface
in a cylindrical tank. Dispersant is applied in a fine stream and then
mixing energy is supplied by a pressurized water stream. The tank con-
tents are recirculated after which samples are withdrawn for extraction
and spectrophotometric analysis.
The standard toxicity test involves exposing three species (Pimephales
promelas, Fundulus heteroclitus, and Artemia salina) to dispersant and
oi1/dispersant mixtures. Prom these tests a curve relating organism
survival to material concentrations is developed to determine median
tolerance limits.
Separate discussion sections include the statistical analyses of "tes-
ting the test" results for reproducibility and the rationale for se-
lecting the test procedures as presented.
111
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ACKNOWLEDGEMENTS
The authors wish to convey their appreciation to those who contributed
to the development of this report.
Special acknowledgement is given to T. A. Murphy, Ph.D., formerly Chief,
Oil Spills Research Branch, Edison Water Quality Research Laboratory,
and C. M. Tarzwell, formerly Director, National Marine Water Quality
Laboratory, Narragansett, Rhode Island who directed the initial research
efforts which developed the standard dispersant tests.
Valuable assistance 1n the preparation of the final manuscript and re-
view of the contract reports were furnished by R. J. NadeauKPh.D.,
biologist, and T. Roush, biologist, of the Oil Spills Research Branch,
Edison Water Quality Research Laboratory.
The author is grateful to Marion Curry, Writer-Editor, Technical Infor-
mation Office, NERC, Cincinnati for her review of the original manuscript.
The serveral drafts and final manuscript were typed by Ann Krypel, whose
patience and forbearance is gratefully appreciated.
1v
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CONTENTS
Section Page
I INTRODUCTION 1
Perspective 3
Background 5
II EPA STANDARD DISPERSANT TEST PROCEDURES 9
EPA STANDARD DISPERSANT EFFECTIVENESS TEST 11
Scope and Application 11
Definition 11
Summary of Method 11
Apparatus 11
Reagents 13
Oils 14
Dispersant Dosages 15
Replications 15
Preparation of Stock Oil Solutions 15
Preparation of Standard Curves 15
Procedure 16
Calculations 18
Test Results 20
EPA STANDARD DISPERSANT TOXICITY TEST 22
Scope and Application 22
Definition 22
Summary of Method 22
Measures of Toxicity 22
Selection and Preparation of Test Materials 22
Preparation of Experimental Water 24
Standard Freshwater Formulation 24
Standard Seawater Formulation for Fish 24
Standard Seawater Formulation for Artemia 25
Sampling and Storage of Test Materials 25
No. 2 Fuel Oil 26
General Test Conditions and Procedures for
Bioassay 27
Dissolved Oxygen and Aeration 27
Transfer of Organisms 28
Test Duration and Observations 28
Physical and Chemical Determinations 29
Testing Laboratory 29
Test Containers 29
Preparation of Test Concentrations 31
Calculations and Reporting 32
Summary of Procedures 33
BIOCHEMICAL OXYGEN DEMAND TEST 35
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Section Page
III DISCUSSION OF DISPERSANT EFFECTIVENESS TEST 37
Statistical Analysis 39
Comment on Test Revisions 42
IV DISCUSSION OF TOXICITY TEST 51
Statistical Analysis 53
Comment on Test Revisions 53
REFERENCES 57
vi
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FIGURES
Page
1. Test Tank 12
2. Suggested Hosing System 12
3. Schematic Diagram of Automatic Dispensing 30
Pipette System
4. Mean and Confidence Limits (95%) of 48 Hour 54
TL50 Artemia salina Bioassay Results for
Three Laboratories
5. Mean and Confidence Limits (95%) of 96 Hour 54
TL5Q Fundulus heteroclitus Bioassay Results
for Three Laboratories
vii
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TABLES
No. Page
1 Unit Costs vs. Cleanup Costs 4
2 Properties of Test Oils 7
3 Composition of Synthetic Sea Water 13
4 Characteristics of Test Oils 14
5 Dispersant Blank Correction Factors 19
6 Required Dispersant Effectiveness Test Results 21
7 Standard Seawater Formulation for Fish 24
8 Standard Seawater Formulation for Artemia 25
9 Characteristics for No. 2 Fuel Oil 26
10 Analysis of Variance 40
11 Analysis of Variation 41
12 Sources of Variation and Percent of Total
Variation within 3x4x4 Factorial Design 55
viii
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SECTION I
INTRODUCTION
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INTRODUCTION
Perspecti ve
Several hundred products are currently available for the purpose of
emulsifying or dispersing oil slicks (1). The products are marketed
and sold throughout the country to individuals who have little or no
knowledge of their relative effectiveness and toxicity. This lack of
awareness is due, in part, to the unavailability of information con-
cerning the relative advantages and disadvantages of specific disper-
sants, and this may result in erroneous conclusions and expensive er-
rors in judgment.
From the consumer's point of view, the least expensive product, on a
cost per gallon basis, may not necessarily be the most economical. For
instance, although product A (Table 1) is less expensive on a unit cost
basis than product B, it Is less efficient, a.nd therefore more expen-
sive to use. From the environmentalists' points of view, the problem
of product selection requires consideration of the toxicity, as well as
the efficiency, of the dispersant being compared. It would be futile
to use a particular dispersant whose toxicity was 10 times less than all
other available dispersants if 10 times more material were required to
disperse the same given quantity of oil. Additionally, dispersant ef-
fectiveness and toxicity must be known to realistically estimate the
volume required for storage and cleanup;-:
Thus, the need to determine relative dispersant effectiveness and toxi-
city is clear. The U.S. Environmental Protection Agency (EPA) has
satisfied this need according to the schedule of events that will be
described shortly.
It should be emphasized at this time that the EPA Standard Dispersant
Effectiveness and Toxicity Tests presented in this report are laboratory
procedures only. The provide a means for conducting comparisons among
diverse products manufactured to accomplish a single goal - the disper-
sing of oil slicks. These products are sold, without formula modifica-
tions, for use under extremely variable environmental conditions. To
develop a test that would compare hundreds of products under all possi-
ble conditions would be impossible. Yet, the comparisons must be made.
The dispersant tests presented in this report provide a reasonable
compromise suitable for comparative laboratory testing of relative
effectiveness and toxicity.
The results of the effectiveness test, therefore, should not be con-
sidered directly applicable to actual situations. The d«ta are for
comparison purposes only. Likewise, the toxicity test described in
this report represents artificial conditions. It is expected, how-
ever, that the relative order of the dispersant-effectiveness and toxi-
city-test results would be applicable to field situations.
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TABLE 1. UNIT COSTS COMPARED WITH CLEANUP COSTS
Ci. . ... Dispersant
Stipulation ^—c g-
Volume of oil (gal) 100 100
Recommended application
ratio (d1spersant/o1l) 1:5 1:10
Required volume of
dispersant (gal) 20 10
Costs of dispersants
($/gal) 3 4
Cleanup costs, total $ 60 40
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Replication of a test organism's natural environment is extremely diffi-
cult, and thus use of a natural environment was sacrificed because of
the need for constant and defined test conditions. Therefore, the data
generated from toxicity tests should not be considered directly eco-
logically relevant. The test was not designed to provide data that
could be used to directly assess the potential effect of dispersants on
the biota of an aquatic system; rather, it was to provide a standard
toxicity value (TLso) that would be useful for comparing a dispersant's
toxicity against known standards and other dispersant products. The
consumer will then know that the toxicity and effectiveness values
quoted by the manufacturer are comparable and reproducible in the la-
boratory with the use of standard procedures.
Background
In October 1968, the Federal Water Quality Administration (FWQA), a
predecessor of the U.S. Environmental Protection Agency, developed and
issued a policy statement on the "Use of Chemicals to Treat Oil Spills."
This policy was submitted to and subsequently approved by the Secretary
of the Department of the Interior, the agency under which FWQA then
operated. This policy represented the first major step taken by a
regulatory agency toward the rational use of chemicals, including dis-
persants, for the protection of the environment from oil pollution. It
directed the Administration to develop a standard laboratory procedure
for determining the acute toxicity of such chemicals, specifically dis-
persants. An Interim Toxicity Procedure was developed and subsequently
published on April 28, 1969. Further improvements and modifications of
these tests, which were later expanded to include the development of pro-
cedures for measuring dispersant emulsion efficiency and stability, were
continued until July 19, 1969, when the procedures for toxicity and
emulsion efficiency were submitted to the Assistant Secretary, U.S. De-
partment of the Interior, for approval.
The Assistant Secretary deemed industry's acceptance of these standard
procedures to be critical. The procedures were therefore reviewed for
technical accuracy and scientific significance by an Ad Hoc Committee
consisting of representatives from industry, state and water pollution
control regulatory agencies, and the academic community.
Following the first American Petroleum Institute/Federal Water Pollution
Control Administration Joint Conference on Prevention and Control of Oil
Spills, held in December 1969, the Ad Hoc Committee convened and, after
two days of discussion accepted the two procedures presented, with only
minor technical modifications. The Committee did recommend, however,
that both laboratory procedures be considered as "Tentative Assay Meth-
ods" until they had been evaluated for precision and accuracy by labora-
tories outside the Federal government.
Following this recommendation, bids for "testing the tests" were soli-
cited and research contracts awarded (in April 1970) to:
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Syracuse University Research Corporation
Life Sciences Division
Syracuse, New York 13210
Pacific Environmental Laboratory
657 Howard Street
San Francisco, California 94105
University of Miami
School of Marine and Atmospheric Sciences
10 Rickenbacker Causeway
Miami s.Florida 33149
New England Aquarium
Central Wharf
Boston, Massachusetts 02210
Two of the laboratories (Pacific Environmental Laboratory and New Eng-
land Aquarium) evaluated both the toxicity and emulsion efficiency pro-
cedures; the other two laboratories concentrated on only one of the
procedures (Syracuse University Research Corporation - dispersent ef-
ficiency; University of Miami - dispersant toxicity.) In addition, all
laboratories were asked to determine the biodegradability and ultimate
and 5-day biochemical oxygen demand (BOD), of selected dispersants,
using standard accepted procedures.
Data generated by the investigating laboratories are evaluated in this
report. The recommended tests for measuring efficiency and acute toxi-
city of oil spill dispersants are presented and discussed.
The four laboratories participating in the study were instructed to fol-
low the procedures presented in "Standard Tests for Measuring Oil Dis-
persant Toxicity and Emulsion Efficiency" (2). These procedures were
published in the "Proceedings of the Joint Conference on Prevention and
Control of Oil Spills," December 1969 (3 and 4). The revised procedures
are presented in this report.
Four test oils were investigated during the course of the study: South
Louisiana Crude Oil, Bachequero Crude Oil, Number Two Fuel Oil and Number
Six Fuel Oil (Table 2). These oils were selected on the basis of their
relative availability, wide range of physical and chemical properties,
and high degree of spill incidents.
Four commercially available dispersants, representing the generic range
of products available, were also studied:
A - water based, nonionic polyhydric alcohol ester of
fatty acid.
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TABLE 2. PROPERTIES OF TEST OILS
Property
Gravity, APP
Viscosity, SUS
G> 1000F
Pour point, °F
Sulfur weight, %
Asphaltenes weight, %
Neutralization no.
Distillation
volume distilled, %
9 300° F
@ 500°F
So. Louisiana
Crude
36.6
41
15
0.20
0.3
0.40
18
43
Bachaquero
Crude
17
1500
- 5
2.2
7.0
2.7
6
17
No. 2
Fuel Oil
32
35
-20
0.3
0.05
5
50
No. 6
Fuel Oil
10
4500
60
2.5
10.0
2.5
0
5
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B - oil based, blend of anionic alky! aryl benzene
and nonionic wetting agents.
C - water based, nonionic blend of alkanol amides
D - oil based, nonionic emulsifier in petroleum
based solvents.
The test oils, dispersants, test tank for dispersant effectiveness, and
related equipment were supplied to the contracting laboratories by the
Edison Water Quality Research Laboratory.
The military procurement specifications for oil slick solvent emulsifier,
MIL-S-22864A (SHIPS) of 24 February, 1969, was used as a basis for de-
veloping the test for dispersant effectiveness described here.
The bioassay methods for determining the short-term toxicity of various
materials to fishes described in Standard Methods for the Examination of
Water and Wastewater" (5) were the basis for the tests described herein
for determining the relative toxicity of dispersants and similar oil
spill treatment products to aquatic organisms. Because of particular
problems arising in the bioassay of oil and oil dispersants mixtures and
the need for comparable results, certain procedures described in Stan-
dard Methods have been made more specific. The changes include the
addition of special bioassay methods or organisms other than fish and
the standardization of certain elements and conditions of the test such
as the test species, mixing and agitation, dilution water, aeration,
dissolved oxygen (DO) concentrations, temperatures, pH, and salinity.
Throughout the test period, attention was given to those aspects of the
procedures that could be modified, tightened, or otherwise improved.
The recommended modifications incorporated into the revised standard
tests are discussed in detail in subsequent sections of this report.
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SECTION II
EPA STANDARD DISPERSANT TEST PROCEDURES
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EPA STANDARD DISPERSANT EFFECTIVENESS TEST
Scope and Application
This method applies to all substances defined as Dispersing Agents (or
Dispersants) by the National Oil and Hazardous Substances Pollution
Contingency Plan (August 1971), Annex X (6). This is the EPA standard
dispersant effectiveness procedure required in Annex X of the Contin-
gency Plan.
Definition
For the purpose of this method, the definition of Dispersing Agents
(or Dispersant) in Annex X is applicable and is as follows: "Disper-
sing Agents are those compounds which emulsify, disperse or solubilize
oil into the water column or act to further the surface spreading of oil
slicks in order to facilitate dispersal of oil into the water column".
Summary of Method
Oil is applied to the surface of synthetic sea water contained in a
cylindrical tank. The dispersant is applied in a fine stream to the oil
and a 1.0 minute contact time is allowed for the dispersant to contact
the oil. Energy is imparted to the oil/dispersant mixture by hosing and
agitating the mixture with a pressurized water stream. The contents of
the tank are allowed to recirculate and samples are withdrawn from the
tank after 10 minutes and after 2 hours of recirculation. Oil is ex-
tracted from the samples with chloroform and the quantity of oil is
spectrophotometrically determined.
Apparatus
1. Test Tank: Construct test tank, 24 inches, ID, by 28 inches high
of 16-gauge stainless steel and equipment, as shown in Figure 1,
with associated piping, valve, and pump for recirculation of dis-
persed oil and for sample collection.
2. Oil Containment Ring: Use 16-gauge stainless steel ring, 7% inches
in diameter and 9 inches in length to contain the oil while the oil
contacts the dispersant. Fit the ring with clamps so that it can be
placed vertically in the center of the test tank with its midpoint
16 inches above the base of the tank. Design the clamps so the ring
can be quickly removed from and replaced in the tank.
3. Hosing System: Provide a pressurized hosing system suitable for de-
livering synthetic sea water to the oil/dispersant mixture in the
test tank. A suggested hosing system is shown in Figure 2. Deliver
hosing water through a 1/2-inch ID hose, which is connected to a
shut-off nozzle consisting of a cast brass ball shut-off valve with
11
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3/4 I.D PVC PIPING (RIGID)
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a 3/16-inch ID discharge tip. (Akron Brass Company, Style 111
shut-off valve with Style 558, 3/16-inch tip, or equal).
To obtain a 20 psig nozzle flow rate (the specified pressure during
the hosing operation) hose synthetic sea water at 23 +_ 1°C into a
calibrated container for a predetermined time. This rate shall be
4.0 j^0.2 gpm @ 20 psig. Use a suitable valve in the hose line to
adjust and control the hosing pressure. The pressure should be
determined by means of a pressure gauge in the line immediately
before the nozzle or by a pitot gauge reading in the synthetic sea
water stream.
Corrosion buildup within the nozzle may change hosing pressure and
alter test results. To prevent this, remove and flush the nozzle
with fresh water at the end of each day's tests.
4. Spectrophotometer: Use a spectrophotometer suitable for measurement
at 340 my and 620 my to photometrically determine oil concentration
of the oil/chloroform mixture. A Bausch and Lomb Spectronic 20
spectrophotometer (or equal) is acceptable for this purpose.
5. Dispersant Dispensing Bottle: Use a 250-ml-capacity dispensing
bottle with a 3-mm orifice tip to apply dispersant to the oil in
the test tank. A Fisher Scientific Company Nalgene bottle (Catalog
No. 3-409-15-A) or equivalent with its fine tip cut off to expose a
3-mm opening is acceptable for this purpose.
5. Filter Paper: Use a filter paper suitable for filtering the oil/
chloroform extract. Whatman No. 1 filter paper (or equivalent) is
acceptable for this purpose.
Reagents
1. Synthetic Sea Water: To prepare synthetic sea water solutions,
dissolve the amounts of Reagent Grade chemical salts shown in Table
3 in water having a total hardness of less than 50 mg/1:
TABLE 3. COMPOSITION OF SYNTHETIC SEA WATER
Salt
NaCl
MgCl2-6H20
Na2S04
CaCl2
KC1
NaHC03
A
g/i
17.096
7.444
2.852
0.802
0.483
0.140
B
or g/100 gal
6,471
2,931
1,080
304
183
53
If any salt other than those listed above are used, allowances must
be made for water of crystal!ation.
13
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2.
3.
After dissolving the salts and thorough aeration, the resultant
solution should be clear, with a pH between 7.9 and 8.3, and a
salinity of 25.00 + 0.50%.
If necessary, adjust pH to 8.0 +0.1 with HC1 or NaOH.
Use this salt solution for initial test tank contents and for hosing
during test procedure.
Note: For convenience, a concentrated solution in an amount
sufficient for 10 tests (exclusive of hosing) may be prepared
by dissolving 3.13 times the quantity of salts listed in (B)
above per 100 gallons. Ten gallons of this concentrated solu-
tion diluted to the 16-inch level in the test tank will pro-
vide the required salt concentration for testing.
Chloroform, Reagent Grade
Sodium Sulfate, Anhydrous, Reagent Grade
Oils
Test the dispersant with 100 ml of No. 2 Fuel Oil and No. 6 Fuel Oil.
These oils shall have characteristics as defined in Table 4.
TABLE 4. CHARACTERISTICS OF TEST OILS
Characteristic
No. 2 Fuel Oil
Min. Max.
No. 6 Fuel Oil
Min. Max.
Gravity
Viscosity
Kinematic ®100°F
Furol e 122°F
Flash point
Pour point
Cloud point
Sulfur
Aniline point
Carbon residue
Water
Sediment
Ash
Aroma tics
Distillation
IBP
10%
50%
90%
End point
Aphaltenes
Neutralization No.
°API 32.1
(cs) 2.35
42.8
3.00
(SFS)
(°F) 150
( F
(°F1
(wt %)
(°F) 125
(Wt %)
(Vol %
(Wt %
(Wt %
-
-
-
(Vol %) 10
(°F) 347
(°F) 402
(°F) 475
(°F) 542
(°F) 596
0
+10
0.35
180
0.16
0
0
-
15
407
456
530
606
655
(Wt %)
-
0.05
8.9 16.9
-
101 250
160
+35
-
2.73
-
12.3
0.20
0.10
0.10
-
10.0
2.5
14
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Dispersant Dosages
Test the dispersant at the following dosages:
10 ml, 25 ml, 50 ml, and 100 ml.
Replications
Replicate each test a minimum of five (5) times.
Preparation of Stock Oil Solution
Dissolve exactly 3.250 grams of test oil 1n 1 liter of chloroform in a
1-liter volumetric flask and mix well.
(1 ml = 3.25 mg of oil)
Preparation of Standard Curves
(Suggested procedure when utilizing Bausch and Lomb Spectronic 20
spectrophotometer)
No. 2 Fuel Oil: Prepare the standard curve from the stock solution as
follows:
Dilute 10 ml, 25, ml, and 50 ml of stock solution to
100 ml with chloroform in 100-ml volumetric flasks.
Mix well.
Determine spectrophotometrically the Optical Density (O.D)
of the stock solution and the diluted aliquots in a 1-inch
cell at a wavelength of 340 my.
Plot the standard curve as milligrams of oil versus O.D.
No. 6 Fuel Oil: Prepare the standard curve from the stock solution as
follows:
Dilute 10 ml, 25 ml, 40 ml and 50 ml of stock solution
to 100 ml with chloroform in 100 ml volumetric flasks.
Mix well.
Determine spectrophotometrically the O.D. of the diluted
aliquots in a 1/2-inch cell at a wavelength of 620 my.
Plot the standard curve as milligrams of oil versus O.D.
15
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Procedure
Step 1: Fill the test tank to a height of 16.0 inches with synthetic
sea water at 23 +_ l°c.
Step 2: Clamp the oil containment ring to the test tank. The ring
shall be positioned in the center of the tank with its mid-
point 16 inches above the base of the tank.
Step 3: Slowly add 100 ml of the test oil at 23 +_ IOC from a 100-ml
graduated cylinder with a rapid circular motion onto the
water surface within the center of the ring. Care should be
exerted so that oil is not lost below the ring and that oil
does not contact the ring walls upon application.
Allow the oil to drain from the graduated cylinder for 3.0
minutes after application.
Weigh the graduated cylinder before and after application to
determine the exact amount of oil used in the test. Record
weights to 0.1 gram for use in final calculations.
Step 4: Add the dispersant at 23 +_ 1°C onto the oil surface within the
ring in a fine stream from the dispersant dispensing bottle,
using minimum force to maintain flow of dispersant.
Add the dispersant with a rapid circular and criss-cross motion.
Carefully apply the dispersant onto the oil surface only and
not through the oil surface or onto the ring walls.
Apply the dispersant during a time period of less than 2
minutes.
Weigh the dispersant dispensing bottle before and after appli-
cation to determine the exact amount of dispersant used in
the test. Record weights to 0.1 gram for use in final cal-
culations.
Step 5: After completely applying the dispersant, allow an additional
1.0 minute contact time before hosing.
Step 6: Activate the hosing system, adjust nozzle pressure to 20 psig,
and apply a stream of synthetic sea water at 2.? +_ 1°C to the
oil/dispersant mixture within the ring. Immediately lift the
ring out of the test tank, and simultaneously hose off the
oil adhering to its inner surface. Remove the ring completely
and continue to hose and agitate the oil/dispersant mixture
for a total hosing period of 1.0 minute. (Flow rate of hosing
nozzle must be 4.0 +_ 0.2 gpm at 20 psig.)
16
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Note: (1) Removing the ring should take no longer
than 5 to 10 seconds.
(2) To hose the oil/dispersant mixture, hold
the discharge tip of the nozzle approximately
level with the top edge of the test tank and
pointed vertically downward. Move the nozzle
rapidly in a random manner from side to side,
backwards and forwards, and around the inner
wall of the tank, as necessary, to facilitate
continuous hosing and agitation of the entire
oil/dispersant surface.
Step 7: Immediately after hosing, start the recirculation pump and
continue recirculation for 2.0 hours.
Step 8: After 10.0 minutes of recirculation, withdraw a 500-ml sample
into a 500-ml graduate cylinder and discard. Immediately
collect another 500-ml sample for determining "initial dis-
persion."
Step 9: After 2.0 hours of recirculation, withdraw a 500-ml sample in-
to a 500-ml graduate cylinder and discard. Immediately col-
lect another 500-ml sample for determining "final dispersion".
Step 10: Transfer the 500-ml sample to a 1-liter separatory funnel.
Add 25 ml of chloroform to the separatory funnel, stopper
funnel, and shake vigorously for 50 strokes.
After shaking, place the funnel in a rack, vent, and allow
a settling time of 2 to 3 minutes.
After settling period, lift the funnel from the rack and
gently invert several times. While holding the funnel, allow
contents to settle and then gently swirl with a circular mo-
tion to afford additional settling of the oil/chloroform
mixture.
Transfer the oil/chloroform mixture to a 250-ml Erlenmeyer
flask that contains anhydrous Na^SO. for drying the extract.
Repeat the extractions using a total of at least three, 25 ml
portions of chloroform.
Step 11: After the oil extraction is complete, filter the combined ex-
tracts from the Erlenmeyer flask through dry filter paper into
an appropriate size volumetric flask (100 ml, 250 ml, or 500 ml
depending on the amount of chloroform used to complete the
extraction).
17
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Rinse the ^$04 and filter paper with small portions of
chloroform to remove entrained oil.
After rinsing, fill the volumetric flask to the mark with
chloroform, stopper, invert, and thoroughly mix contents.
Spectrophotometrically determine the O.D. of the extract
using the identical wavelength and cell established for the
test oils. From the standard curve for the test oil, deter-
mine the quantity of oil in milligrams.
Step 12: Blank Correction Determination
Perform test with 100 ml dispersant in the absence of oil with
the following modifications:
(a) Add dispersant from a 100-ml graduate cylinder
(calibrated to contain 100 ml) into synthetic sea
water in test tank without the oil containment ring.
(b) Allow the dispersant to drain from the graduated
cylinder for 3.0 minutes after application.
(c) Using 50 ml of synthetic sea water, rinse the
dispersant from the graduate into the tank. Repeat
rinsing two times.
Continue test procedure Step 6 through Step 11. The total volume of
dispersant/chloroform extract shall not exceed 100 ml.
Spectrophotometrically determine the O.D. of the extract using the iden-
tical wavelength and cell established for the test oils. From the stan-
dard curve for each test oil, determine the dispersant interference
expressed as milligrams of oil. This quantity of oil, referred to as "X
in the table under Calculations, is the blank correction for 100 ml dis-
persant in 100 ml total extract.
Additional blank corrections for other test conditions can be determined
by multiplying "X" by the appropriate factors indicated in the table.
Calculations
UU II
1. Dilution Factor (DF): The dilution factor is 1, 2.5, or 5 depending
upon whether the total volume of oil/chloroform extract was 100 ml,
250 ml, or 500 ml, respectively.
2. Oil Conversion Factor (OCF): Calculate the OCF as follows:
OCF = Sp Gr of oil x 100 ml
grams of test oil applied
18
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3. Dispersant Conversion Factors (DCF): Calculate the DCF as follows:
For 10 ml dispersant:
Sp Gr of dispersant x 10 ml
grams of dispersant applied
For 25 ml dispersant:
_ Sp Gr of dispersant x 25 ml
grams of dispersant applied
For 50 ml dispersant:
= Sp Gr of dispersant x 50 ^ml
grams of dispersant applied
For 100 ml dispersant:
= Sp Gr of dispersant x 100 ml
grams of dispersant applied
The specific gravity @ 60/60F should be determined to +_ 0.0001.
4. Blank Correction (BC): Calculate BC (expressed as milligram of oil)
for the dispersant as outlined in Table 5.
TABLE 5. DISPERSANT BLANK CORRECTION FACTORS
Volume of test
dispersant
100 ml
50 ml
25 ml
10 ml
Blank correction (mg
Volume of chloroform
100 ml
X*
0.50X
0.25X
0.10X
250 ml
0.40X
0.20X
0.10X
0.04X
of oil)
extract
500 ml
0.20X
0.10X
0.05X
0.02X
*Refer to Step 12 of Procedure for determination of "X."
5. Mi Hi grams of Oil Equivalent to 100% Dispersion: Complete disper-
sion (100%) of 100 ml of test oil in the testing procedure is equi-
valent to 0.749 ml of oil/liter of sample after completion of tank
test procedure.
Convert the above volume of oil (0.749 ml/liter) to milligram of oil
per 500 ml sample (the quantity of sample required in the test pro-
cedure) as follows:
19
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Milligrams of oil equivalent to 100% dispersion =
500 ml sample x 0.749 ml «. r - n., 1000 mg
1000 ml samplex bp br OT U1' x gram
375 x Sp Gr of oil
This result, expressed in milligrams, is equivalent to 100% disper-
sion of 100 ml of test oil per 500 ml sample after completion of
tank testing procedure.
Percent Dispersion: Calculate the percent dispersion as follows:
Percent dispersion =
(mg oil from standard curve x DF x OCF xDCF) - BC x 100
mg oil equivalent to 1001 dispersion
Test Results
1. Based on 100 ml of oil, determine the percent dispersion of each of
the two test oils caused by 10, 25, 50, and 100 ml of dispersant:
(a) after 10 minutes recirculation ("initial dispersion");
and
(b) after 2 hours recirculation ("final dispersion").
2. Determine the mean of at least 5 replicate tests for each of the
four dispersant dosages. If the percent dispersion value found
(after the 10-minute recirculation period only) for any of the 5
replicate tests varies from the mean value by more than +^8, dis-
card that result and run another replicate.
3. For each test oil, using percent dispersion as the ordinate and dis-
persant dosage (ml) as the abscissa, plot two graphs, one for "ini-
tial dispersion" and the other for "final dispersion." Draw the
graphs by plotting mean percent dispersion values for each of the
dispersant dosages (10, 25, 50, and 100 ml) and connecting in a
straight line fashion each of the data points. From the "initial
dispersion" graph, determine the dispersant dosage (ml) causing 50%
dispersion. From the "final dispersion" graph, determine the dis-
persant dosage (ml) causing 25% dispersion.
4. When reporting test results, provide the following information for
each of the two test oils:
20
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TABLE 6. REQUIRED DISPERSANT EFFECTIVENESS TESTS RESULTS
Volume
Dispersant,
ml
10
25
50
100
Initial dispersion (10 minutes)
% Dispersion
for replicate 1
1 -
2 -
3 -
4 -
5 -
1 -
2 -
3 -
4 -
5 -
1 -
2 -
3 -
4 -
5 -
1 -
2 -
3 -
4 -
5 -
Mean %
dispersion
-
-
-
-
Dosage (ml) causing 50% dis-
persion (from "initial disper-
sion" graph)
ml
Final dispersion (2 hours)
% Dispersion
for replicate 1
1 -
2 -
3 -
4 -
5 -
1 -
2 -
3 -
4 -
5 -
1 -
2 -
3 -
4 -
5 - '
1 -
2 -
3 -
4 -
5 -
Mean %
dispersion
-
-
-
-
Dosage (ml) causing 25%
dispersion (from "final
dispersion" graph)
ml
21
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EPA STANDARD DISPERSANT TOXICITY TEST
Scope and Application
This method applies to all substances defined as Dispersing Agents (or
Dispersants) by the National Oil and Hazardous Substances Pollution
Contingency Plan (August 1971), Annex X (6). This is the EPA standard
dispersant toxicity procedure required in Annex X of the Contingency
Plan.
Definition
For the purpose of this method, the definition of Dispersing Agents (or
Dispersants) in Annex X is applicable and is as follows: "Dispersing
Agents are those compounds which emulsify, disperse or solubilize oil
into the water column or act to further the surface spreading of oil
slicks in order to facilitate dispersal of oil into the water column."
Summary of Method
The standard toxicity test for dispersants involves exposing three
species (Pimephales promelas, Fundulus heteroclitus, and Artemia salina)
to five different concentrationsof_each dispersant alone and injnix-
tujjjd±b-JJiej*e^gjiinended te^J~01T]7?iai^T"o~of 1:10_(djispersint~to oil)
From these testsTa~curve relaTThg to the survival of tfie organisms to
the concentrations of the test material can be generated that allows the
determination of median tolerance limits. To ensure consistency in the
assays carried out by different workers and to detect any changes in the
condition of the test organisms that might lead to different results,
reference bioassays will be made with doceyl sodium sulfate in addition
to the usual control tests.
Measures of Toxicity
Express bioassay results in terms of tolerance limits (TL). Along with
the symbol TL, the time of exposure and the percentage of fish surviving
are indicated. For example, a 96-hour TLso of a toxic substance is that
concentration in which 50% of the fish survive for 96 hours. The
is equivalent to the median tolerance limits (TL5o).
Determine the 96-hour TLso f°r the fish bioassays and 48-hour TLso for
the Artemia bioassays.
Selection and Preparation of Test Materials
1. Test Organisms: The test organisms will be: freshwater-fathead min-
now (Pimephales promelas); saltwater-mummichog (Fundulus hetero-
clitus); and brine shrimp (Artemia salina).
22
-------
Preparation of Test Organisms: Obtain test fish from a common
source for any one series of bioassays. Report any known unusual
condition to which fish were exposed before use (e.g., pesticides
or chemotherapeutic agents); avoid if possible. Use small fish
preferably 1 to 1-1/2 inches in length and weighing about 1 gram.
The longest individual should be no more than 1.5 times the length
of the smallest.
Acclimate test fish for use in the bioassays to the selected tempera-
ture and salinity for a period of 10-14 days before being used for
the bioassays to determine relative toxicity. Disregard groups of
fishes having more than 20% mortality during the first 48 hours and
more than 5% thereafter. During acclimation, feed all species a
balanced diet. Dry, pelleted, commercially available fishfood con-
taining 30-45% protein is satisfactory. The protein source will be
fishmeal, alone or with other animal protein. The pellets should be
of a size for ready consumption by the test fish. Feed the fish
twice daily to satiation, but not for 48 hours before or during the
bioassay test. Use only those organisms that feed actively and ap-
pear to be healthy. Discard any individuals injured or dropped
while handling. During acclimation of marine species, keep water
temperatures at a final level of 20 +_ 1°C; pH at a level of 8.0
with a variation of j^U.l unit; and salinity at a level of 20 +_ 0.5
ppt. Acclimate freshwater species to a water temperature of 25 +_
PC and a pH of 7.5 to 7.8. Keep oxygen levels above 4 ppm, pre-
ferably between 5-6 during the acclimation period. Run all tests
in a series with organisms from the same group.
To ensure uniformity of Artemia, secure the eggs from the San Fran-
cisco Bay area. Since the eggs of Artemia may be kept dessicated
for long periods in a viable state, required numbers of the organ-
ism can be secured at any time for use in the bioassay tests through
the use of proper hatching procedures.
A rectangular tray (plastic, glass, or enamel) having 200 square
inches of bottom surface is suitable for hatching Artemia eggs. Di-
vide this tray into two parts by a partition that extends from the
top down to about 0.75 to 0.5 inch from the bottom. This partition
may be of any opague, biologically inert material (a pasteboard
strip, sealed with paraffin wrapping, is satisfactory.) Raise one
end of the tray about 0.5 inch and add 3 liters of the standard sea-
water formulation (See Table 8.) Spread 0.5 gram of brine shrimp
eggs in the shallow end of the tray. Cover this end of the tray with
a piece of cardboard to keep the eggs in darkness until hatching is
complete. About 20 hours after the eggs hatch, direct a narrow beam
of light across the uncovered portion of the tray. Since the brine
shrimp are phototactic, they will swim beneath the partition into
the illuminated end of the chamber and congregate in the beam of
light. The Artemia concentrated in the beam of light can be easily
collected with the use of a collecting pipette or siphon connected
23
-------
to a 12-inch rubber tube and mouthpiece. Transfer them to a beaker
having a small amount of the artificial seawater.
Preparation of Experimental Water
Because large quantities of dilution water will be used in these tests,
formulate the experimental water in large batches to ensure uniformity
and constant condition for the various tests. To prevent contamina-
tion, prepare and store the experimental water in fiber glass containers
of suitable size.
Standard Freshwater Formulation
Prepare the standard freshwater as follows: bubble C02 through 10 liters
of distilled water containing 2.1 gram of CaCOo to convert it to a clear
solution of Ca(HC03)2- Dilute this solution with 20 liters of distil-
led water and add 30 ml of a mixed salt solution. To prepare this mixed
salt solution dissolve 23.4 grams of NaCl, 73.9 grams MgSO^HgO and
4.4 grams 1^504 in distilled water and dilute to 1 liter.
Use any multiple of these volumes and weights to secure desired volumes
of the standard water. This water is very close in dissolved materials
to the average natural stream water of the United States. When pre-
paring the standard freshwater, use distilled water from a glass still
or from a tin-lined still to ensure the absence of metal ions. The pH
of this water shall be in the range of 7.6 to 7.8; to adjust, add or
remove C02-
Standard Seawater Formulation for Marine Fish
To prepare standard seawater for marine fish, mix technical grade salts
with 900 liters of distilled or demineralized water in the order and
quantities listed in Table 7. These ingredients must be added in the
order listed and each ingredient must be dissolved before another is
added. Sitr constantly during preparation to accomplish this.
TABLE 7.
STANDARD SEAWATER FORMULATION FOR MARINE FISH
Ingredient Amount, grams Ingredient
SrCl2.6H20*
H3B03
KBr
CaCl2-2H20
20
30
100
1100
MgCl2-6H20
NaCl
Na2Si03*9H20
EDTA -tetra sodium salt*
Amount, grams
5000
23500
20
3
*Reagent grade if technical grade not available.
**Ethylene diamine tetracetate.
24
-------
Add distilled or demineralized water to make up to 1000 liters. The
pH should now be 8.0 +_ 0.1. To attain the desired salinity of 20 +_
0.5 ppt at time of use dilute with distilled or demineralized water.
Standard Seawater Formulation for Artemia
The standard seawater used in bioassays with Artemia is prepared in the
same manner and is essentially the same water used in the fish bioassays.
Because much smaller quantities are needed, the formulation in Table 8
is suggested.
To formulate this water, mix technical grade salts with distilled or de-
mineralized water in the order and quantities listed in Table 8, Dilute
the final water with distilled or demineralized water to achieve a sa-
linity of 22 ppt. If necessary, add NaHC(h to adjust final pH of water
to between 8.0 and 8.2. Before the water is used, filter it through a
0.22 micron Mi Hi pore filter under vacuum.
TABLE 8. STANDARD SEAWATER FORMULATION FOR ARTEMIA
Ingredient Amount, grams Ingredient Amount, grams
SrCl2-6H20
H-BO,,
3 3
KBr
CaCl2-2H20
Na2S04
0.02
0.03
0.10
1.10
4.00
MgCl2-6H20
NaCl
Na2Si03-9H20
EDTA**
10.00
23.50
0.02
0.003
*Ethylene diamine tetracetate
Ingredients are dissolved in 900 ml of water in order given and
solution is diluted to 1 liter.
Sampling and Storage of Test Materials
Store oil used in bioassays in sealed containers to prevent the loss of
volatiles and other changes. For ease in handling and use, it is recom-
mended that 100 ml glass containers be used and a minimum of 40 con-
tainers of each grade or kind of oil under test be available. 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 comple-
tion of the dosing to prevent its use at a later date when it has lost
its volatiles. Run all tests in a bioassay series with oil from the
same container and with organisms from the same group collected or se-
cured from the same source.
25
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No. 2 Fuel Oil
Perform the dispersant bioassays with No. 2 Fuel Oil having the char-
acteristics defined in Table 9.
TABLE 9. CHARACTERISTICS OF NO. 2 FUEL OIL
Characteristic
Gravity
Viscosity
Kinematic @ 100°F
Furol $ 122°F
Flash point
Pour point
Cloud point
Sulfur
Aniline point
Carbon residue
Water
Sediment
Ash
Aroma tics
Distillation
IBP
10%
50%
90%
End point
Aphaltenes
Minimum
°API 32.1
(cs) 2.35
(SFS)
(°F) 150
(°F)
(°F)
(Wt 3!)
(°F) 125
(Wt 35)
(Vol 35)
(Wt %)
(Wt 35)
(Vol %) 10
(°F) 347
(°F) 402
(°F) 475
(?F) 542
(°F) 596
(Wt %)
Maximum
42.8
3.00
-
0
+10
0.35
180
0.16
0
0
-
15
407
456
530
606
655
-
Neutralization no. - 0.05
26
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General Test Conditions and Procedures for Bioassay
Temperature: For these bioassays, use test solutions with temperatures
of 25 +_ 1°C for freshwater species and 20 +_ PC for salt water species.
Dissolved Oxygen and Aeration
1. Fish: Because oils and dispersants contain volatile materials, many
of which are toxic 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.
Initiate aeration to provide DO and mixing after the fish are added.
The DO content of test solutions must not drop below 4 ppm. To
limit the removal of volatiles, keep aeration to a minimum: aerate
only enough to keep the DO concentration about 4 ppm. Aerate at a
rate of 100 j^ 15 bubbles per minute supplied from a 1-ml serological
pipette having a 1-mm ID. At this rate and with the proper weight
of fish, DO concentration can be maintained slightly above 4 ppm
over a 96-hour period. Take DO measurements at least daily.
2. Artemia: The surface area of the test solution exposed in relation
to volume is so large that DO is not a problem. Oxygen content re-
mains high throughout the assay because of the small quantity of
test substances added and the low oxygen demand of organisms in
each dish.
3. Controls: With each fish or Artemia test or each series of simul-
taneous 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 experi-
mental water or diluent alone as the medium in which the controls
are held. There must be no more than 10% mortality among the con-
trols during the course of any valid test and at least 90% must re-
main apparently healthy.
To ensure consistency in the assays carried out by different workers
and to detect changes in the condition of the test organisms that
might lead to different results, perform reference bioassays made
with reagent grade dodecyl sodium sulfate (DSS) commonly known as
sodium lauryl sulfate in addition to the usual control tests. To
prepare a stock solution of DSS with either the fresh or salt water
formula, immediately before use add 1 gram of DOS per 500 ml solu-
tion. When all conditions of the bioassay are met and the outlined
tests are carried out, results of bioassays made in different areas
and by different investigators will be comparable and serve to indi-
cate the relative toxicity of different dispersants and oil/disper-
sant mixtures. 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.
27
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4. Number of Organisms: For the bioassay procedures utilizing fish
place two fish in each jar. For the bioassay utilizing Artemia,
place 20 larvae in each container.
Transfer of Organisms
Transfer fish from the acclimatizing aquaria to the test containers
only with small-mesh dip nets of soft material and do not rest on any
dry surface. Do not hold out of the water longer than necessary. Dis-
card any specimen accidentally dropped or otherwise mishandled during
transfer.
Artemia can be conveniently handled and transferred with a small pipette
connected to a 12-inch rubber tubing and mouthpiece. To have the neces-
sary Artemia ready for the study, transfer 20 Artemia into small beakers
containing 20 ml of artificial seawater. Hold these batches of Artemi a
until they are 24 hours old; at that time, place them in the respective
series of test concentrations set up for the bioassay.
To avoid large fluctuations in the metabolic rate of organisms and the
fouling of test solutions with metabolic waste products and uneaten
food, do not feed organisms during tests.
Test Duration and Observations
1. Fish: 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 varia-
tions in opercular movement, coloration, body orientation, movement,
depth in container, schooling tendencies, and other. Abnormal be-
havior of the test organisms is a desirable parameter to monitor in
a bioassay because changes in behavior and appearance may preceed
mortality. Toxicants can reduce an organism's ability to survive
natural stresses. In these cases, the mortality is not directly at-
tributed to the toxicant, but most certainly is an indirect effect.
Reports on behavioral changes during a bioassay can give insight
into the nonacute affects of the tested material.
At the end of the 96-hour period, terminate the fish assays and
determine the Tl-50 values.
2. Artemia: Terminate the Artemia test after 48 hours of incubation.
To count the dead animals accurately and with relative ease, place
the test dishes on a black surface and hold a narrow beam of light
parallel to the bottom of the dish. Most of the dead Artemia will
28
-------
be on the bottom of the test dish and can be readily seen against
the black background. Also search the top of the liquid for Artemia
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 per-
iodic spasm of its entire body. For this test, animals exhibiting
any movement when touched with a needle are considered alive. Ac-
count for all test animals to ensure accuracy since some Artemia
may disintegrate and lose integrity. Consider individuals not ac-
counted for as dead.
Physical and Chemical Determinations
Determine the temperature, DO, and pH of test solutions and the measur-
able toxicant concentrations before introducing the organism, and as
soon as possible after the death of all or most of the organisms, or at
the end of a test where organisms are still alive. The additional
determinations of temperature and DO are needed for proper control of
test conditions and to facilitate interpretation and application of the
bioassay results, make them at regular intervals.
Testing Laboratory
An ordinary heated or air-conditioned laboratory room with thermostatic
controls suitable to maintain prescribed test temperatures generally
will suffice to conduct the bioassays. Where ambient temperatures can-
not be controlled to the required temperature range, use water baths
with the necessary temperature controls.
Test Containers
Process all required glassware before each assay. 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.
For fish tests, use 4-liter glass jars measuring approximately 22.5 cm
high, 15 cm in diameter, and 11 cm in diameter at the mouth. The jars
are to have screw top lids, lined with aluminum foil. In carrying out
the test, add 2 liters of the standard seawater formulation aerated to
saturation with DO to each of the jars. To add the 2 liters easily and
accurately, use a 2-liter capacity, automatic dispensing pipette (Fig-
ure 3).
For the Artemia tests, use Carolina culture dishes (or their equivalent)
having dimensions approximately 3-1/2 by 1-1/2 inches.
29
-------
00
o
SOO GALLONS
I
\
A=INFIOW FROM LARGE HOLDING RESERVOIR
B = OV£BHOW FROM OTHER UNITS IN SERIES
C=INFLOW TO OTHER UNITS
OVEHflOW
1 GALLON (US)
- 2 LITERS
SCHEMATIC DIAGRAM OF AUTOMATIC DISPENSING PIPETTE SYSTEM
Figure.3
-------
Preparation of Test Concentrations
1. Fish: Place the test jars containing 2 liters of standard water on
a reciprocal shaker. The shaker platform should be adapted to
firmly hold six of the bioassay jars. Add the desired amount of
the petroleum product under test with a calibrated syringe directly
to each test jar. Dispense the appropriate amount of the dispersant-
standard water stock into the jars with a pipette. Tightly cap the
test jars and shake for 5 minutes at approximately 315 to 333 three-
quarter-inch strokes per minute in a reciprocal shaker. At the
completion of shaking, remove the jars from the shaker to a constant-
temperature water bath or room, remove the lids, and add two test
fish.
2. Artemia: To prepare test solutions for dispersants and oil/disper-
sant mixtures: (1) Blend or mix with an electric blender having
speeds of 10,000 to 20,000 rpm or less, a stainless steel cutting
assembly, and 1-liter borosilicate jar. To minimize foaming, blend
at speeds below 10,000 rpm. (2) For the dispersant test solution,
add 550 ml of the standard seawater to the jar, then with the use
of a gas-tight calibrated glass syringe with a Teflon-tipped plun-
ger, add 0.55 ml of the dispersant and mix for 5 seconds. (3) For
the oil/dispersant mixture, add 550 ml of the standard seawater to
the mixing jar. While the blender is in operation, add 0.5 ml of
the oil under study with the use of calibrated syringe with Teflon-
tipped plunger and then 0.05 ml of the dispersant as indicated above.
Blend for 5 seconds after addition of dispersant. These additions
provide test solutions of the dispersant and the oil/dispersant mix-
ture at a concentration of 1000 ppm of dispersant and oil/disper-
sant combination, respectively, and in a 1:10 ratio of dispersant
to oil.
3. Fish and Artemia: Immediately after the test solution of the dis-
persant or oil/dispersant solution is prepared, with the use of a
gas-tight Teflon-tipped glass syringe of appropriate size, draw up
the necessary amount of test solution and dispense into each of the
five containers in each series. If the series of five concentra-
tions 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: 1, 1.8, 3.2, 5.6 and 10 ml.
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.
Use exploratory tests before the full-scale test is set up to deter-
mine the concentration of reference standard to be used in each of
the five different concentrations. After adding the required
31
-------
amounts of liquid bring the volume in each of the test containers
up to 80 ml with the artificial seawater. To ensure keeping each
of the series separate, designate on each lid of the test containers
the date, the material under test, and its concentration.
When the desired concentrations are prepared, gently release into
each dish the 20 test Artemi a (previously transferred into 20 ml
of medium). This provides a volume of 100 ml 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.
After added the test animals, incubate the assay dishes at 20°C for
48 hours. Recommended lighting is 2100 Iumens/m2 (200 ft-c) of
diffused, constant, fluorescent illumination coming from beneath
the culture dishes during incubation. Because Artemi a are photo-
tatic, bottom lighting should keep them from direct contact with
the oil that sometimes layers on top.
Wash the blender thoroughly after each use and repeat the above pro-
cedures for each series of tests. To wash the blender: (1) Pour a
strong solution of laboratory detergent into the blender to cover
the blades. (2) Fill the container about half of its volume with
hot tap-water. (3) Operate the blender for about 30 seconds at
high speed. (4) Remove and rinse twice with hot tap-water, mixing
each rinse for 5 seconds at high speed. (5) Rinse twice with dis-
tilled water, mixing each rinse for 5 seconds at high speed.
Calculations and Reporting
At the end of the test period, the bioassays are terminated and the
values are determined.
1. Calculations: A TLso is a concentration at which 50% of the experi-
mental animals survived, or it is an interpolated value based on
percentages of organisms surviving at two or more concentrations, at
which less than half and more than half survived. Estimating the
TLso by interpolation involves plotting the data on semi logarithmic
coordinate paper with concentrations on the logarithmic axis and
percentage survival on the arithmetic axis. A straight line is
drawn between two points representing survival at the two successive
concentrations that were lethal to more than half and to less than
half of the organisms. The concentration at which the line crosses
the 50% survival line is the Tl_50 value.
2. Reporti ng : The tested dispersant and oil and their source and stor-
age are described in the bioassay report. Note any observed changes
in the experimental water or the test solutions. Also include the
species of fish used, the source, size, and condition of the fish,
data on any known treatment of the fish for disease or infestation
with parasites before their use, and any observations on the fish
32
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behavior during tests. In addition to the calculated USQ values,
data used to make the calculations (i.e., the percent survival at
the end of each day of exposure at each concentrations of toxicant)
must be reported.
Summary of Procedures
Fish:
a. Prepare adequate stocks of the appropriate standard dilution
water.
b. Add 2 liters of the standard dilution water to the 4-liter test
jars. The 5 repetitions of each of 5 dispersant or oil/disper-
sant concentrations will require 35 jars.
c. Add the determined amount (quarter points on the log scale) of
test material to the respective jars with gas-tight, Teflon-
tipped glass syringe.
d. Cap the jars tightly with the aluminum-foil-lined screw caps
and shake for 5 minutes at 315 to 333 three-quarter-inch strokes
per minute on a reciprocal shaker.
e. Remove the jars from the shaker, and add 2 acclimated fish per
jar. Execute these operations at 20 +_ 1°C for marine fish and
25 +_ 1°C for freshwater fish.
f. To maintain oxygen levels above 4 ppm, aerate with 100 + 15
bubbles per minute through a 1 ml serological pipette with an
ID of 1 mm.
g. Observe and record mortalities and behavioral changes each
24 hours.
h. After % hours, terminate the assay, and plot the data on semi-
log graph paper to determine TL5Q.
Artemia
a. Initiate the procedure for hatching the Artemia the day before
the bioassay is to be conducted so that 24-hour-old larvae are
available.
b. With the use of the small pipette, transfer 20 Artenia into
each of 35 small beakers each containing 20 ml of the proper
artificial seawater.
c. To prepare the test stock dispvrsant solution, add 550 ml of
the artificial seawater to tht prescribed blender jar. By
33
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means of a gas-tight glass syringe with a Teflon-tipped plunger,
add 0.55 ml of the dispersant and mix at 10,000 rpm for 5 sec-
onds.
To prepare the test stock oil/dispersant 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 dispersant with the use of a calibrated syringe with a
Teflon-tipped plunger. Blend for 5 seconds after adding the
dispersant. One ml of these stock solutions added to the 100 ml
of standard seawater in the assay containers yields a concen-
tration of 10 ppm dispersant or oil/dispersant combination and
the latter will be in a ratio of 1 part dispersant to 10 parts
of oi1.
d. Each test consists of 5 replications of each of 5 concentrations
of the material under study, a control series of 5 dishes, and
a standard reference series of 5 different concentrations, a
total of 35.
Immediately after preparing the test solution of the dispersant
or oil/dispersant solution, and using an appropriately sized
syringe, draw up the necessary amount of test solution and dis-
pense 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 opera-
tion. Turn mixer off immediately after the sample is taken to
limit the loss of volatiles.
After adding the required amounts of the test oil/dispersant or
dispersant mixture, bring the volume of liquid in each of the
test containers up to 80 ml with the artificial seawater.
When the desired concentrations are prepared, gently release in-
to each dish the 20 test animals previously transferred into
20 ml of medium. This provides a volume of 100 ml 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.
e. Wash the blender as prescribed for each series of tests.
f. Incubate the assay dishes at 20 + 1°C for 48 hours with the pre-
scribed lighting.
g. Terminate the experiment after 48 hours, observe and record the
mortalities, and determine the TLcn as prescribed.
34
-------
BIOCHEMICAL OXYGEN DEMAND TEST
jf
Since biodegradability or oxygen demand factors have a substantial
bearing on the selection of a particular chemical material to treat
oil floating on water, 5-day BOD data on dispersants must be submitted
to EPA. ., -;.
The procedure to be used for this determination is found in:
Standard Methods for the Examination of Water
and Wastewater, 13th Edition, p. 489, Method
219 (1971),
or
ASTM Standards, Part 23, Water; Atmospheric
Analysis, p. 712, Method D - 2329-68 (1970).
35
-------
SECTION III
DISCUSSION OF DISPERSANT EFFECTIVENESS TEST
37
-------
DISCUSSION OF DISPERSANT EFFECTIVENESS TEST
Statistical Analysis
According to test requirements, each laboratory conducted at least three
replicate series of tests on each of the 16 oil/dispersant combinations.
Additional testing, as procedural modifications were incorporated into
the test routine, produced a total of over 380 analyses from each labor-
atory. Several procedural discrepancies, attributable to the preliminary
nature of the original test procedure, were discovered, however, when
the results from all laboratories were compared. Therefore, a complete
statistical analysis could not be performed on the entire set of data.
The analysis of variance of the standard percent dispersion for 10 ml of
dispersant, one of several data points common to all laboratories which
could be statistically examined, is presented in Table 10. The 10 ml
point was chosen for analysis because it provided a realistic indication
of the degree of reproducibility among laboratories and is one of the
points required in the revised test procedure.
A four by four nested factorial design was used to determine that part
of the total variation attributable to each of the four major experi-
mental variables represented in Tables 10 and 11 by the following sym-
bols:
0 - Oils (4 types)
L = Laboratories (3 contracts)
D = Dispersants (4 products)
R = Replicates (3 of each test combination)
An F-test was applied to determine if each source of variation was sig-
nificant at the 95% confidence level.
The results of the analysis presented in Table 10 indicate that replica-
tion was involved in every instance where there was no statistical dif-
ference. This is to be expected as replication tends to dampen variation
within test runs. Eliminating replication as a source of variation and
analyzing only the oils, dispersant, laboratories, and their interactions
yields the results presented in Table 11.
Examination of Table 11 reveals that (1) the variations among oils, dis-
persants, laboratories, and their interactions are statistically signi-
ficant 99% of the time; (2) major variation is, as expected, the result
of the four different oils and four different dispersant; and (3) the
variation that results from the laboratories is approximately 11% of the
39
-------
TABLE 10. ANALYSIS OF VARIANCE
Source of
variation
0
L
D
R
OL
00
LD
OR
LR
OR
OLD
OLR
ODR
LDR
OLDR
Sums of
squares
15033
3152
17720
1849
1201
5361
5456
229
189
739
5466
591
562
327
985
Degress of
freedom
3
2
3
2
6
9
6
6
4
6
18
12
18
12
36
Means
squares
5011
1576
5097
924
200
596
909
38
47
123
304
49
31
27
27
% of
total
31.7
9.9
37.4
5.8
1.2
3.7
5.7
2.1
0.2
0.7
1.9
0.3
0.1
0.1
0.1
F°-95= m
87.9
27.7
103.7
16.2
3.5
10.5
16.0
0.7
0.8
2.2
5.3
0.9
0.5
0.5
0.5
Si gni f i cant
di f ference
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
No
No
No
No
40
-------
TABLE 11. ANALYSIS OF VARIATION
Source of
variation
Main effect
0
L
D
Two- factor
interaction
OL
OD
LD
Three -factor
interaction
OLD
Sum
Sum of
squares
15,033
3,152
17,720
1,201
5,361
5,456
5,466
53,389
Degree
of freedom
3
2
3
6
9
6
18
47
Mean
squares
5,011
1,567
5,907
200
596
909
304
14,494
% of
total
34.6
10.8
40.8
1.4
4.1
6.3
2.1
F
0.99
88
28
10.0
3.5
10
16
5.3
Si gni f i cant
di f ference
Yes
Yes
Yes
Yes
Yes
Yes
Yes
41
-------
total variance. This includes the variation due to the procedure, as
well as to inherent laboratory variation resulting from any inconsis-
tencies in equipment performance, ambient temperature, personal habits,
etc. On the whole, an 11% variation is not considered excessive. How-
ever, considerable effort has been expended to tighten the test proce-
dure, and thereby reduce variation and increase reproducibility.
Comments on Test Revisions
The statistical analysis of the dispersant effectiveness test results,
discussed above, indicated that 11% of the total variance in test re-
sults was attributable to differences among the laboratories. A com-
prehensive review of the procedures carried out by each laboratory, as
well as a careful evaluation of the original test method, identified
the need for certain procedural modifications that should significantly
improve both the methodology and the reproducibility of the test, and
consequently, reduce laboratory variation to a minimum.
These revisions include clarification and expansion of critical aspects
of the test not clearly defined in the original procedure, as well as
incorporation of modifications and recommendations suggested by the
laboratories.
The original test procedure was difficult for the reader to follow be-
cause several other documents had been incorporated by reference. All
such cross-references have been eliminated from the revised procedure,
which makes it easier to follow now. The new format should eliminate
some of the misinterpretation associated with the testing requirements
and procedural steps of the original procedure.
Critical testing criteria, i.e., temperature and time, are now clearly
defined in each and every step of the testing procedure where appropri-
ate.
Precautionary statements, particularly in reference to oil and disper-
sant applications and agitation by hosing, as well as instructions more
clearly defining sampling technique, have been inserted. Methods for
calculating the final results, which include detailed instructions for
normalization of the test results, have also been included. Finally,
the importance of testing and correcting for dispersant interference
is emphasized and expanded.
A detailed discussion of the revisions follows.
T- Test Tank: The original schematic of the test tank has been revised
to include the critical dimensions of component parts not specifi-
cally identified in the original test procedure.
Additionally, a clamp has been incorporated on the recirculation re-
turn line from the pump as recommended by one contractor "so that
42
-------
the piping system remains rigid.'1 line contractor noted that the
slight shift in angle of the retur^ line seemed to alter slightly
the circulation of the tank contents from one run to the next.
None of the laboratories indicated any major problem specifically
attributable to the test tank. EPA furnished each of the contrac-
tors identical test tanks so that problems that would have been
expected if each laboratory had fabricated its own test tank were
avoided.
2. Hosing System: The original test procedure was extremely vague con-
cerning the pressurized hosing system. No schematic of the appara-
tus was given, nor were any details of the hosing nozzle and flow
rates provided. Since the energy imparted to the oil/dispersant
mixture is a function of the pressurized system delivering the
water stream, any variation in nozzle characteristics (orifice size,
flow rate, pressure) could seriously influence dispersant effec-
tiveness.
These deficiencies have been corrected by incorporating a sketch of
the hosing system along with a detailed specification for the noz-
zle and its required performance characteristics (pressure/flow
rate).
3. Spectrophotometer: The suitability of a Bausch and Lomb (B&L) Spec-
tronic 20 (or equivalent) instrument for determining oil concentra-
tion in chloroform extracts of test samples is clearly stated in
the revised procedure.
The original procedure omitted guidelines on suitable wavelength
and cell size for spectrophotometrically measuring the concentra-
tions of the test oils. The revised procedure suggests measuring
No. 2 Fuel Oil concentrations at 340 mu in a 1-inch cell and mea-
suring No. 6 Fuel Oil concentrations at 620 my in the 0.5-inch cell.
These general guidelines related to spectrophotometry will be most
beneficial for those testing laboratories not equipped for routine
spectrophotometric analysis but that are otherwise technically com-
petent to fulfill the test requirements.
4. Dispersant Dispensing Bottle: In the original procedure, using a
dispersant dispensing bottle for applying dispersant was purely
optional on the part of the laboratory conducting the tests. In
the interest of standardization, the revised procedure now makes
the utilization of this particular device mandatory.
5. Synthetic Sea Water: The revised formula for synthetic sea water
(SSW) includes the addition of potassium chloride (KC1). The new
formulation now consists of the six major elements (Cl, Na, Mg, S,
Ca, and K) that are present in sea water at concentrations of 1%
43
-------
or greater. Collectively, these elements constitute 93% of the
total elemental composition of sea water.
To minimize the difficulty of SSW preparation and the costs of dis-
tillation or purchase associated with the large volumes of water
required in the testing procedure, the option of using a source
water with a hardness (EDTA) of less than 50 mg/1 is now specified.
Commercial softeners with sufficient capacity to produce treated
water with a hardness significantly less than 50 mg/1 are readily
available at minimum costs.
Additionally, by inserting a softener ahead of a hot water heater,
softened hot water is available to expedite the solution of the
salts required to prepare SSW. Use of a softener particularly ap-
plies to preparing the concentrated SSW, which is a new option of
the revised procedures.
After preparing the concentrated SSW, the proper aliquot of the con-
centrate can be diluted in the test tank by interconnection of the
softened cold and hot water supplies. Most importantly, by inserting
valves for adjusting the flow rate of the two water supplies, little
difficulty should be encountered in maintaining the precise control
of water temperature at 23 +_ 1°C from test to test. The water tem-
perature fluctuations experienced by one laboratory were attributed
to the variability in temperature of the distilled water purchased
from a supplier.
It is anticipated that the emphasis placed on water temperature re-
quirements, together with the optional suggestions relating to water
hardness and concentrated SSW preparation Twill aid the testing la-
boratories in handling the large volumes of water required for tes-
ting.
6. Test Oils: Three important considerations in the development of
standard test procedures are: economy, simplicity, and reproduci-
bility. Each of these played a key role in the selection of oils
for the dispersant effectiveness test.
Crude oils were tested in the original procedures. However, the fol-
lowing problems were encountered: (1) crude oils were difficult to
obtain in gallon or single drum quantities. Refineries, terminals,
and similar facilities that handle crude oils are not equipped to
provide such small volumes; (2) crude oils from the same field vary
significantly in chemical and physical properties. Additionally,
a refinery may handle a particular crude for a short time period
and then switch to another as the supply or demand varies; and (3)
there are over 8,000 different crude oils worldwide. How represen-
tative of this picture one or two local crudes might be is not
known.
44
-------
For these reasons, the revised dinners ant effectiveness i.~5t pro-
cedures do not specify crude oils.'',
The principle characteristics of oils that affect the efficiency of
dispersants are viscosity, surface tension, and density. From ex-
perience and manufacturers' data, some oils are known to be "easy"
to disperse and some are "difficult" to disperse. The revised pro-
cedure specifies No. 2 Fuel Oil, which is relatively easy to dis-
perse, and No. 6 Fuel Oil, which is relatively difficult to disperse.
These oils represent a large percentage of spill incidents and are
readily obtainable. Additionally, it is expected that the range of
physical characteristics of the test oils will generally reflect the
broad range of crude oils having characteristics within the limits
of the test oils.
The original test procedure referenced ASTM specifications for No. 2
and No. 6 Fuel Oils. Yet it is known, for example, that the visco-
sity for No. 6 Fuel Oil, according to ASTM specifications, varies
from 900 to 9000 cs and that this variation will greatly influence
the resultant dispersion. It is also known that ASTM specifications
contain no allowance for the aromatic content of No. 2 Fuel Oil,
which varies from 5% to 40% on a weight basis. In addition to a
tremendous effect on toxicity, this variation could also influence
dispersant effectiveness. With knowledge such as this experience
gained at the Edison Water Quality Research Laboratory, and recom-
mendations from the contract laboratories, the decision was made to
omit the ASTM specifications and publish more precise specifications
on the characteristics of the test oils.
In the revised test procedure, detailed specifications are estab-
lished for No. 2 and No. 6 Fuel Oils. Because these specifications
must be met at the time of testing as well as at the time of pur-
chase, proper preservation, storage, and quality control of the test
oils is required.
7. Dispersant Dosages: The three participating laboratories were re-
quired to determine the dosages of dispersant causing 25%, 50%, and
75% oil dispersion. This procedure turned out to be extremely time
consuming since it required testing by trial and error. Also, for
any oil/dispersant combination, the target percent dispersion points
(25%, 50% and 75%) were never exactly reached by all three labora-
tories. Accordingly, this procedure made it extremely difficult to
determine common data points from which to compare results among the
laboratories.
The revised procedure now requires the preparation of curves that
relate dispersant dosage to percent dispersion. A mere informative
and statistically sound method of measuring percent dispersion at
preselected specific volumes of dispersant (10 ml, 25 ml, 50 ml and
100 ml) is now stipulated.
45
-------
8. Preparing Stock and Standard Solutions of Oil: Suggested procedures
for preparation of stock and standard working test oil solutions are
outlined in the revised test. Although specifically referenced for
application when the B&L Spectronic 20 is used, the format for pre-
paring standard solutions with appropriate volumetric modifications
is generally applicable for use with other spectrophotometers. Like-
wise, the stock solution, prepared as proposed, is applicable for
use regardless of which spectrophotometer is employed.
9. Water Temperature: Presently, the degree to which test-water temp-
erature variations influence dispersant effectiveness is not de-
fined; however, test-water temperature is known to directly effect
the physical characteristics of both the oil and dispersant. The
physical characteristics of the dispersion formed and its subsequent
stability are also dependent on water temperature. Therefore, it is
critical that the test water temperature be precisely controlled.
The water temperatures employed at the three laboratories varied:
Lab 1, 16.QOC to 28.(PC; Lab 2, 20.(PC to 22.20C; and Lab 3, 21.(PC
to 25.0°C.
In the analysis of variance, the extent to which test-water tempera-
ture differences between the laboratories contributed to the total
variance is not known. In the original procedure the intent was to
have the effectiveness tested at a water temperature of 23j^ 2°C.
Unfortunately, this water temperature requirement was not specifi-
cally nor clearly stated in the documents outlining the test pro-
cedure, and two of the three laboratories misinterpreted it. To
overcome the possible recurrence of the problem, the revised test
procedure incorporates water temperature requirement of 23 +_ 1°C in
Step 1. To avoid any misinterpretation, the requirement is addi-
tionally included in Step 6 that relates to hosing with SSW.
The required temperature, which approximates room temperature, is
considered to be a realistic compromise considering the large volume
of water involved in the testing procedure. Most laboratories are
expected to have little difficulty in precisely controlling tempera-
tures at this level. Such precise control is highly desirable when
considering the potentially large number of laboratories reporting
results to EPA.
10. Oil and Dispersant Temperatures: No consideration was given in the
original test procedure to the question of controlling the tempera-
tures of the oils and dispersants. The properties of some oils and
oil/dispersant mixtures (e.g., viscosity, etc.) change radically with
temperature and consequently influence the effectiveness rating of
the dispersant under consideration. To eliminate this influence and
to increase reproducibility of the test, the revised procedure now
provides for maintaining the oil and dispersant at temperatures of
23 + IOC.
46
-------
11. Oil and Dispersant Applications: The technique for applying oil
and dispersant in the original procedures lacked sufficient details
to assure uniformity of applications from test to test. The re-
vised procedure elaborates the mode of application; details are
expanded and precautionary statements are included to avoid cer-
tain techniques that could cause loss of oil and dispersant to the
underlying water or cylinder walls before completion of the oil/
dispersant contact time.
Gravimetric determination of the amounts of oil, as well as disper-
sant, applied in the testing procedure are now required for sub-
sequent normalization of test results.
The revised procedure should make possible the application of more
precise quantities of the required volumes of oils and dispersants
to be tested, as well as minimize the degree of normalization re-
quired for adjusting test results.
12. Contact Time: The laboratories were required to determine whether
the contact time (the period between the addition of dispersant to
the oil and the onset of hosing) influenced dispersant effective-
ness. For each oil/dispersant combination, the percent dispersion
for contact periods of zero (instantaneous) and 10 minutes were
measured. Analysis of the data indicated that the effectiveness
was independent of contact time. Although the laboratories did not
always agree on the "best contact time," the differences in effec-
tiveness found by a given laboratory at zero contact time as com-
pared with 10-minute contact time were not, in most cases, greater
than the differences in effectiveness found by the laboratory be-
tween replicate runs for the same contact time. Therefore, for
the purposes of standardization of the test conditions and for
ease of carrying out the test procedure, the test method has been
revised to require testing at only a single contact time -- 1.0
minute.
13. Hosing Operation: The original test method required the oil/dis-
persant mixture in the test tank to be agitated with a pressurized
hose stream of SSW. This hosing was to be continued until the
water level in the tank increased 2 inches. Because of the turbu-
lence of the tank contents and the foaming and frothing of the oil/
dispersant mixtures during hosing, determining when the 2-inch
level had been reached and exactly when to terminate hosing, was
extremely difficult. Consequently, data from the laboratories in-
dicated that the time of hosing varied from 40 to 90 seconds. Any
variation in hosing alters the amount of agitation of the oil/dis-
persant mixtures, which consequently affects oil dispersion. To
eliminate the time and volumetric errors inherent in this hosing op-
eration, the procedure was modified by establishing a volume flow
rate for the hosing stream (4.0± 0.2 gpm at 20 psig) and then desig-
nating a specific hosing period for the operation — 1.0 minute.
47
-------
14. Sampling Techniques: The original test required recirculation of
the contents of the test tank for a predetermined period at which
time a 500 ml sample was immediately taken. One of the contract
laboratories indicated that when the valve was opened to remove the
sample, water with a high oil content was nearly always detected in
the volume collected from the first few seconds of flow. It was
stated that the probable reason was that a trap was formed by the
short dead-end line leading to the spigot in which the oil concen-
trated during the recirculation period.
To eliminate the above effect and ensure a representative sample,
the revised procedure now requires purging the recirculated stream
by discarding the first 500 ml sample and collecting a subsequent
sample for analysis.
15. Sampling Times: The original test required the collection of a
sample for initial dispersion after 5 minutes recirculation. It
also required sampling for dispersion stability after 15 minutes
30 minutes, and 1, 2, 4, and 6 hours of recirculation. A review
of the stability data furnished by the three contract laboratories
indicates that the instability of any particular dispersion will
normally be manifested within 2 hours; therefore, any testing be-
yond this time frame should not be necessary. Therefore, for ease
and simplicity of testing, the original number of samplings has
been reduced to only two, i.e., 10 minutes for "initial dispersion"
and 2 hours for "final dispersion.".
16. Dispersant Blank Correction : Although the original test required
checking for dispersant interference in the absence of oil and cor-
recting for this interference when appropriate, one laboratory
failed to run the blank tests. Another laboratory ran the blank
tests but failed to correct results for the two dispersants pro-
ducing positive and significant interference.
Two laboratories failed to properly interpret the requirements for
blank corrections. To avoid recurrence of this problem, a more de-
tailed explanation of the dispersant blank interference testing and
blank correction is outlined in the revised procedure. Also in-
cluded in the revised procedure is a detailed sequential procedure
for calculating test results.
17. Normalization of Results: Significant variations resulted in many
cases because the prescribed method failed to require calculating
a correction factor for the actual quantities of the oils and dis-
persants used in the tests. Since it is difficult to deliver ex-
actly the same quantitites of dispersants and oils from test to
test, the results generated from each test should be adjusted, by
a ratio of preselected quantities of dispersants and oil, for the
quantity of dispersant and oil actually used in the particular test.
For example, the revised procedure requires testing at 10, 25, 50,
48
-------
and 100 ml of dispersant with 100 ml oil. Accordingly, the test
method now requires adjustment or normalization of the results by
multiplying each result by a "dispersant conversion factor"
10, 25, 50, or 100 ml dispersant
ml dispersant used
and by an "oil conversion factor"
100 ml oil
ml oil used
This normalization of the data will reduce the results to a com-
mon end point and should tend to improve the accuracy and precision
of the test.
49
-------
SECTION IV
DISCUSSION OF TOXICITY TEST
-------
DISCUSSION OF TOXICITY TEST
Statistical Analysis
The degree of reproducibility of bioassay results obtained from the
testing laboratories was a major determinent in assessing the validity
of the test procedure and the appropriateness of each species as a test
organism. To assess the degree of agreement among laboratory results,
the bioassay values from each laboratory were calculated and plotted,
with 95% Confidence Limits (Figures 4 and 5.) Overlapping Confidence
Limits indicate that the differences are not significant.
The Least Significant Interval test, plus the percent variation infor-
mation in Table 12, were the basis for evaluating the interim toxicity
test procedure designed for each test species.
The Artemia bioassay exhibited the greatest "among laboratory" varia-
tion with 22% of the total variation attributable to the laboratories.
Figure 4 illustrates this large variation: the mean 48-hour TLso for one
laboratory is significantly different from the other two laboratories.
Close examination of all the data reveals that toxicity values for one
particular oil/dispersant combination were considerably different from
the results for the other oil/dispersant combinations.
For both fish species, the total variation attributable to laboratories
was less than 13%. A situation similar to that of the Artemia assay
was evident for the Fundulus bioassay (Figure 5), however. The mean
96-hour TLso f°r one laboratory's bioassay results was significantly
greater than that for the other two laboratories. In both instances,
the same oil/dispersant combination was responsible for the large vari-
ation in the reported values. This discontinuity in the data demon-
strates the need to blend the test dispersant and oil sufficiently.
Statistical analysis of the bioassays data indicates that less than 22%
of the total variation is attributable to differences among laboratories.
The variation associated with each laboratory results from differences
in such factors as laboratory personnel, preparation of materials, and
source of organisms. Discrepancies arising from these factors can, how-
ever, be reduced by carefully scrutinizing and upgrading each bioassay
procedure. The following discussion presents that qualifications and
alterations to the test procedure that should enhance the reproduci-
bility of the tests, lessen the variation attributed to laboratories,
and, thereby, lend more credibility to the data.
Comments of Test Revisions
1. Test Oils: The standard test oil, No. 2 Fuel Oil, was chosen for
its known relative toxicity, ease of acquisition, and consistency of
composition as compared with crude oils and other petroleum pro-
ducts, and the ease of preparation for testing. For example, high
53
-------
SO •
40 •
DISPEftSANT/Oll IATIO = I 10
PACIFIC
EMGIAMD
MEAN AND CONFIDENCE LIMITS (95%) OF 48 HOUR Tlso
Artemio solino BIOASSAY RESULTS FOR THREE LABORATORIES
Figure 4
70O •
DliPEISANT/Oll IATIO = 1:10
JOO
ZOO
IOO •
MIAMI
PACIFIC
NEW fNGlAND
MEAN AND CONFIDENCE LIMITS (95%) OF 96 HOUR TL$0
h«t«foclifu» BIOASSAY RESULTS FOR THREE LABORATORIES
Figure 5
54
-------
TABLE 12. SOURCES OF VARIATION AND PERCENT OF TOTAL
VARIATION WITHIN 3x4x4 FACTORIAL DESIGN*
Sources
Laboratories
Oil/dispersant
combination (B)
Interaction (AxB)
Dispersants (C)
Interaction (AC)
Interaction (BC)
Residual (ABC)
Brine shrimp
Artemia
21.6
8.1
9.1
22.3
21.8
8.4
8.3
Fat-head minnow
Pimephales
7.6
40.0
4.0
24.2
5.5
14.8
3.8
Munnrichog
Fundul us
12.8
13.8
14.1
16.0
14.4
14.9
13.7
*Bioassay results from oil/dispersant mixtures combined
in a ratio of 1:10.
55
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viscosity oils are difficult to draw into the Teflon-tipped
syringes, to measure, and to extrude into the blender vessel.
Oyster Larvae Bioassay: The oyster larvae bioassay procedure dis-
cussed by Tarzwell (3) was omitted from the standard toxicity test
because of the inability of many laboratories to spawn oysters and
rear early-stage larvae. At best, oyster culturing is tenuous and
the successful laboratories usually specialize only in bivalve
rearing. Acquiring sexually mature oysters on a year round basis
is a difficult task, and a stock of several mature adults would be
necessary to produce enough larvae to run bioassays. Another pro-
blem is inducing oysters to spawn. In addition, producing viable,
healthy oyster larvae is difficult because of the naturally high
mortality of the early stages.
Because oyster larvae have high commerical value, are ecologically
important, and have a known susceptibility to toxicants, they are
an ideal species for testing; however, the problems associated with
maintaining, spawning, and rearing osyters are too complicated to
include their use in the standard dispersant toxicity tests.
56
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REFERENCES
1. Anon. 1971. Oil Spill Dlspersant Product Data, EPA, Edison Water
Quality Laboratory, Edison, New Jersey.
2. Anon. 1969. Standard Tests for Measuring Oil Dlspersant Toxicity
and Emulsion Efficiency, Unpublished. U.S. Dept. of Int., FWPCA.
3. Tarzwell, C. M. 1969. Standard Methods for the Determination of
Relative Toxicity of Oil Dispersants and Mixtures of Dispersants
and Various Oils to Aquatic Organisms. In Proc. Joint Conference
on Prevention and Control of Oil Spills, API and FWPCA, December
15 - 17, 1969, New York, pp. 179-186.
4. Murphy, T. A., McCarthy, L. T. 1969. Evaluation of the Effective-
ness of Oil Dispersing Chemicals. In Proc. Joint Conference on Pre-
vention and Control of Oil Spills, API and FWPCA, December 15 - 17,
1969, New York, pp. 199-207.
5. Standard Methods for the Examination of Water and Wastewaters, 1969.
12th Edition American Public Health Assoc., New York.
6. National Oil and Hazardous Substances Pollution Contingency Plan,
Council on Environmental Quality. August 1971.
57
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
i. Rv^ott No.
w
STANDARD DISPERSANT EFFECTIVENESS AND TOXICITY
TESTS,
McCarthy, Jr., L.T. , Wilder, I., and Dorrler, J. S.
U.S. Environmental Protection Agency
Edison Water Quality Research Laboratory
National Environmental Research Center
Edison. New Jersey. ,0881 7
'2. Si'oasoria. Org»ai'3tiom
5. Report Date
8. 1 rtoTa}i:.xOrgtii..i*tioa
Repot * No.
In-House Project
/.' Type f Repo-.-1
Period Covered
Environmental Protection Agency report
number EPA-R2-73-201 , May 1973
A brief history of the development of the Standard EPA Dispersant Effec-
tiveness and Toxicity tests is outlined. The standard tests are pre-
sented and discussed. An analysis of variance is performed on the data
developed by three independent laboratories in order to determine the
reproducibility of standard test procedures.
In the standard effectiveness test, oil is applied to the water surface
in a cylindrical tank. Dispersant is applied in a fine stream and then
mixing energy is supplied by a pressurized water stream. The tank con-
tents are recirculated after which samples are withdrawn for extraction
and spectrophotometric analysis.
The standard toxicity test involves exposing three species (Pimephales
promelas, Fundulus heteroclitus, and Artemia salina) to dispersant and
oil/dispersant mixtures.From these tests a curve relating organism
survival to material concentrations is developed to determine median
tolerance limits.
Separate discussion sections include the statistical analyses of "tes-
ting the test" results for reproducibility and the rationale for se-
lecting the test procedures as presented.
*0il Dispersants, *Standard Tests, *Bioassay, *Toxicity Test, Emulsifiers
7k !J,r-
Dispersant tests, Dispersant effectiveness tests, Dispersant toxicity test
v • "c:Jjt SecuritrClus.
(Report)
•7. Se<. >rity Ci $s.
1. ' ""!' in i ii. . , U
"27. Ho. ot *
Pay**
.2.' Pr- .a
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U S DEPARTMENT OF THE INTERIOR
WASHINGTON D C 2OZ4O
_ _
Leo T. McCarthy. Jr.
Edison Water Quality Laboratory. NERC
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