2.4.2.3 If one or more of the standard oil extracts do not meet this linear-stability criterion, then the "offending" standard(s) can be prepared a second time (i.e., extraction of the specified amount of oil from 30-ml or seawater for the "offending" standard according to the pretest preparation procedure). If replacement of the reanalyzed standard solution(s) in the standard curve meets the linear-stability criterion (i.e., no RF >20% different from the overall mean), then analysis of sample extracts can begin.

2.4.2.4 If the initial-stability criterion is still not satisfied, analysis of sample extract cannot begin and the source of the problem (e.g., preparation protocol for the oil standards, spectrophotometer stability, etc.) must be corrected.

2.4.2.5 The initial six-point calibration of the UV-visible spectrophotometer at the oil concentrations identified is required at least once per test day.

2.5 Test procedure. 2.5.1 Preparation of premixed dispersant oil. Prepare a premixed dispersant oil by mixing 1 part dispersant to 10 parts oil. Store this mixture in a glass container. The dispersant effectiveness test procedures are listed in steps 1-20:

1. Prepare 4 replicates (same test oil and dispersant), one control (i.e., no dispersant), and one method blank and run at the same time on the shaker table.

2. Add 120±2 ml of synthetic seawater to each of the modified 125-ml glass Erlenmeyer flasks. Measure and record the water temperature.

3. Place the flasks securely into the attached slot on the shaker table.

4. Carefully add 100 μl of an oil-dispersant solution onto the center of the water's surface using a positive displacement pipette.

5. Agitate the flasks for 2011 minutes at 150±10 rpm on the shaker table.

6. After the 2011 minutes shaking, remove the flasks from the shaker table and allow them to remain stationary for 10±1 minutes for oil droplet "settling."

7. At the conclusion of the 10-minute settling period, carefully decant a 30-ml sample through the side spout of the test flasks into a 50-ml graduated cylinder.

NOTE: Discard the first 1-2 ml of sample water to remove nonhomogeneous water-oil initially contained in the spout.

8. Transfer the samples from the graduated cylinder into a 125- or 250-ml glass separatory funnel fitted with a Teflon stopcock.

9. Add 5 ml of pesticide-quality DCM to the separatory funnel and shake vigorously for 15 seconds. Release the pressure carefully from the separatory funnel through the stopcock into a fume hood.

10. Allow the funnel to remain in a stationary position for 2 minutes to allow phase-separation of the water and DCM.

11. Drain the DCM layer from the separatory funnel into a glass-stoppered, 25-ml graduated glass cylinder.

12. Repeat the DCM-extraction process two additional times.

13. Combine the three extracts in the graduated cylinder and adjust the final volume to 20-ml with additional DCM.

14. Analyze the samples using a UV-spectrophotometer at 340, 370, and 400 nm-wavelengths and determine the quantity of oil as follows: (2)

Cx=(Ax)x(RFx)x(VDCM)X(Vtw/Vew)

where: C=Total mass of dispersed oil in swirling flask at wavelength x (x=340, 370, or 400 nm)

Ax-Spectrophotometric absorbance at wavelength x

RF-Mean response factor at wavelength x (determined from equation 1) VDCM-Final volume of DCM-extract of water sample (20 ml)

Vtw-Total water volume in swirling flask vessel (120 ml)

Vew-Volume of water extracted for dispersed oil content (30 ml)

15. Obtain three concentration values for oil in each experimental water sample (340, 370, and 400 nm).

16. Determine the mean of three values as follows:

NOTE: Means will be used for all dispersionperformance calculations. Samples where one of the values for C340, C370, or C400 is more than 30% different from Cmean will be flagged. Whenever oil measurements are flagged as having a concentration based on one wavelength as >30% different from Cmean, raw data will be evaluated to establish that the measurements are valid. In addition, attempts will be made to correlate the difference to oil type, dispersant test, or dispersant used. If no errors or correlations are apparent and >10% of all oil measurements are flagged, the mean concentration data will be used in the calculation for dispersant performance and the subject data will be flagged.

17. Determine the dispersant performance (i.e., percent of oil that is dispersed, or EFF) based on the ratio of oil dispersed in the test system to the total oil added to the system as follows:

EFF (in %)=(Cmean/CTOT)x100 where:

(4)

Cmean Mean value for total mass of dispersed oil in the swirling flask determined by spectrophotometric analysis

CTOT Total mass of oil initially added to the experimental swirling flask

18. Calculate EFF using equation 4 for coupled experiments with and without dispersant (EFF and EFFa, respectively). EFF. is the effectiveness of the control and represents natural dispersion of the oil in the

EFFD=% dispersed oil due to dispersant only EFF=% dispersed oil with dispersant added EFF % dispersed oil with no dispersant added

20. Calculate the average dispersant effectiveness value by summing the corrected values (EFFD) for each of the four replicates for each of the two test oils and dividing this sum by eight.

2.6 Performance criterion. The dispersant product tested will remain in consideration for addition to the NCP Product Schedule if the average dispersant effectiveness, as calculated in section 2.5 above, is at least 45% (i.e., 50%±5%).

2.7 Quality Control (QC) procedures for measurements of oil concentrations. 2.7.1 UVvisible spectrophotometric measurements. At least of

5% all UV-visible spectrophotometric measurements will be performed in duplicate as a QC check on the analytical measurement method. The absorbance values for the duplicates should agree within ±5% of their mean value.

2.7.2 Method blanks. Analytical method blanks involve an analysis of seawater blanks (i.e., seawater but no oil or dispersant in a swirling flask vessel) through testing and analytical procedures (3, pp 79-80). Method blanks are analyzed with a frequency of at least 1 for every 12 experimental swirling flask samples. Oil concentrations in method blanks must be <5% of that occurring for 100% dispersion of oil in testing apparatus.

3.0 Revised standard dispersant toxicity test 3.1 Summary of method. The standard toxicity test for dispersants and other products involves exposing two species (Menidia beryllina (silversides) and Mysidopsis bahia (mysid shrimp)) to five concentrations of the test product and No. 2 fuel oil alone and in a 1:10 mixture of product to oil. To aid in comparing results from assays performed by different workers, reference toxicity tests are conducted using dodecyl sodium sulfate (DSS) as a reference toxicant. The test length is 96 hours for Menidia and 48 hours for Mysidopsis. LCso s are calculated based on mortality data at the end of the exposure period (for method of calculation, see section 3.6 below).

3.2 Selection and preparation of test materials.

3.2.1 Test organisms.

3.2.1.1 Menidia beryllina. Obtain fish (silversides) from a single source for each series of toxicity tests. In-house cultures are

recommended wherever it is cost-effective; however, organisms are available from commercial suppliers. Information on the source of test organisms and any known unusual condition to which fish were exposed before use should be included in the data report. Use of animals previously treated with pesticides or chemotherapeutic agents should be avoided. Organisms should not be used if they appear to be unhealthy, discolored, or show signs of stress. Use 7-day old larval fish. Fish should be cultured in accordance with the methods outlined in Middaugh, et al. (5). There should be no need to acclimate organisms to the 25±1 °C temperature recommended for the toxicity tests if laboratory stock cultures of Menidia are maintained at the recommended culture temperature of 25±1 °C. If test organisms must be obtained from a commercial source, it may become necessary to acclimate test fish to the test temperature of 25±1 °C, a pH of 8.0±0.2, and 2012 ppt salinity since changes in temperature may occur during shipping. Eliminate groups of fish having a mortality of more than 10% during the first 48 hours, and more than 5% thereafter. During acclimation, organisms should be maintained on a diet of freshly hatched Artemia (brine shrimp) nauplii. Feed the fish daily to satiation during the acclimation period, and once daily during the 96-hour test. Care should be taken daily to remove excess food and fecal material from beakers during the test. Use only those organisms that feed actively and that appear to be healthy. Organisms should be free of disease, external parasites, and any signs of physical damage or stress. Discard any fish injured or dropped while handling.

3.2.1.2 Mysidopsis bahia. Several methods for culturing Mysidopsis bahia (mysid shrimp) may be used and are noted in appendix A of Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms (6). To ensure uniformity of mysids, recently hatched mysids should be collected daily from stock cultures and identified by the date of hatch. Mysids used in 48-hour tests should be from a single day's collection, but may have an age range of 5-7 days old. In cases where in-house cultures of mysids are unavailable, organisms may be purchased from a commercial source. Information on the source of test organisms should be submitted in the data report.

3.2.2 Preparation of experimental water. Filtered natural seawater is recommended for use since it represents a natural source of saltwater containing an inherent population of microorganisms. Synthetic seawater formulated according to the following method can serve as an acceptable alternative to filtered, natural seawater for toxicity tests performed in laboratories in which natural seawater is unavailable.

3.2.3 Synthetic seawater formation. To prepare standard seawater, mix technical-grade salts with 900 liters of distilled or demineralized water in the order and quantities listed in table 4. These ingredients must be added in the order listed and each ingredient must be dissolved before another is added. Stir constantly after each addition during preparation until dissolution is complete. Add distilled or demineralized water to make up to 1,000 liters. The pH should now be 8.0±0.2. To attain the desired salinity of 2011 ppt, dilute again with distilled demineralized water at time of use.

or

3.3 Sampling and storage of test materials. Toxicity tests are performed with No. 2 fuel oil having the characteristics defined in table 5. Store oil used for toxicity tests in sealed containers to prevent the loss of volatiles and other changes. For ease in handling and use, it is recommended that 1,000ml glass containers be used. To ensure comparable results in the bioassay tests, use oils packaged and sealed at the source. Dispose of unused oil in each open container on completion of dosing to prevent its use at a later date when it may have lost some of its volatile components. Run all tests in a bioassay series with oil from the same container and with organisms from the same group collected or secured from the same source.

TABLE 4-SYNTHETIC SEAWATER

must not drop below 60% saturation during the first 48 hours of a static acute (96-hour) test and must remain between 40-100% after the first 48 hours of the test. Aeration at a rate of 100±15 bubbles per minute is supplied by a serological pipette as needed for maintenance of DO. If aeration is necessary, all test chambers should be aerated. At this rate, and with the proper weight of fish, DO concentration should remain slightly above 4 ppm over a 96-hour period. Take DO measurements daily.

1 Amount added to 900 liters of water, as described in the text.

2 Ethylenediaminetetraacetate tetrasodium salt.

3.4 General test conditions and procedures for toxicity tests.

3.4.1 Temperature. For these toxicity tests, use test solutions with temperatures of 25±1 °C.

3.4.2 Dissolved oxygen and aeration.

3.4.2.1 Menidia. Because oils contain toxic, volatile materials, and because the toxicity of some water-soluble fractions of oil and degradation products are changed by oxidation, special care must be used in the oxygenation of test solutions. Aeration during the test is generally not recommended but should be used to maintain the required dissolved oxygen (DO) in cases where low DO is observed. The DO content of test solutions

3.4.2.2 Mysidopsis. Achieve sufficient DO by ensuring that the surface area to volume ratio of the test solution exposed is large enough. Oxygen content should remain high throughout the test because of the low oxygen demand of the organisms. Aeration is not recommended during 48-hour acute toxicity tests unless the DO falls below 60% saturation.

3.4.3 Controls. With each fish or mysid test or each series of simultaneous tests of different solutions, perform a concurrent control test in exactly the same manner as the other tests and under the conditions prescribed or selected for those tests. Use the diluent water alone as the medium in which the controls are held. There must be no more than 10% mortality among the controls during the course of any valid test.

3.4.4 Reference toxicant. To aid in comparing results from tests performed by different workers and to detect changes in the condition of the test organisms that might lead to different results, perform reference toxicity tests with reagent grade DSS in addition to the usual control tests. Prepare a stock solution of DSS immediately before use by adding 1 gram of DSS per 500 ml of test water solution. Use exploratory tests before the full scale tests are begun to determine the amount of reference standard to be used in each of the five different concentrations.

3.4.5 Number of organisms. At a minimum, 20 organisms of a given species are exposed for each test concentration. For the toxicity test procedures using Menidia, place 10 fish in each of two jars. For the toxicity tests using Mysidopsis, place 10 larvae in each of two containers.

Organisms

3.4.6 Transfer of organisms. should be handled as little as possible in order to minimize stress. Transfer Menidia and Mysidopsis from the acclimatization aquaria to the test chambers with a pipette or a wide-bore, smooth glass tube (4 to 8 mm internal diameter) fitted with a rubber bulb. Dip nets should be avoided when handling larval fish and mysids. Do not hold fish out of the water longer than necessary and discard any specimen accidentally dropped or otherwise mishandled during transfer.

3.4.6.1 Mysidopsis. To have the mysids ready for study, mysids may be sorted 24 hours prior to initiation of the 48-hour test. Transfer the mysids to a beaker containing a small volume of water; this vessel serves as a holding chamber during randomized transfer of the organisms to test solutions. Mysids are randomly selected from the batch of mysids in the holding chamber, and transferred to 50-ml beakers containing a small volume of seawater. One mysid is added per beaker using a small piece of flexible 500-μm screening until all of the beakers contain one mysid. The process of random selection and sorting is continued until the appropriate number of mysids has been delivered to each of the 50-ml beakers. The mysids are gently released from the 50-ml beakers into larger beakers filled with an appropriate volume of 20-ppt seawater (25 °C) to bring the total volume to 200 ml. The beakers are randomly placed into a temperature-controlled water bath to acclimate overnight at 25 °C. The mysids are transferred to larger beakers (1-liter) for the 48-hour test after the addition of 800 ml of the test solution. A total of 10 mysids per beaker are used for 48-hour acute toxicity tests. A minimum of two replicate chambers are used for each test concentration and control.

3.4.6.2 Menidia and Mysidopsis are fed 50 brine shrimp nauplii/organism daily during the 96-hour and 48-hour tests. Excess food should be removed daily by aspirating with a pipette.

3.4.7 Test duration and observations. 3.4.7.1 Menidia. Observe the number of dead fish in each test container and record at the end of each 24-hour period. Fish are considered dead upon cessation of respiratory and all other overt movements, whether spontaneous or in response to mild mechanical prodding. Remove dead fish as soon as observed. Also note and report when the behavior of test fish deviates from that of control fish. Such behavioral changes would include variations in opercular movement, coloration, body orientation, movement, depth in container,

schooling tendencies, and others. Abnormal behavior of the test organisms (especially during the first 24 hours) is a desirable parameter to monitor in a toxicity test because changes in behavior and appearance may precede mortality. Toxicants can reduce an organism's ability to survive natural stresses. In these cases, the mortality is not directly attributed to the toxicant, but most certainly is an indirect effect. Reports on behavioral changes during a toxicity test can give insight into the non-acute effects of the tested material. At the end of the 96-hour period, terminate the fish tests and determine the LCso values. The acute toxicity test is terminated after four days of exposure. The number of surviving fish are counted and recorded for each chamber in accordance with standard EPA methods (6). The LC50 is calculated using survival data from the test in accordance with the methods described in the guidelines (6).

3.4.7.2 Mysidopsis. Terminate the mysid test after 48 hours of incubation. To count the dead animals accurately, place the exposure vessels on a light table such that light passes through the bottom of the vessel. Most of the dead mysids will be on the bottom of the beaker and can readily be seen against the background of the light table. Also search the top of the liquid for mysids trapped there by surface tension. Exercise caution when determining death of the animals. Occasionally, an animal appears dead, but closer observation shows slight movement of an appendage or a periodic spasm of its entire body. For these tests, animals exhibiting any movement when touched with a pipette tip are considered alive. Account for all test animals to ensure accuracy since Mysidopsis bahia may disintegrate or be cannibalized by other mysids. Consider individuals not accounted for as dead. At the end of 48 hours of exposure, terminate the mysid assay and determine the LC50 values in accordance with the methods described in the guidelines (6).

3.4.8 Physical and chemical determinations. 3.4.8.1 Menidia. Determine the temperature, DO, and pH of the test solutions before the fish are added and at 24-, 48-, 72-, and 96-hour exposure intervals. It is necessary to take measurements from only one of the replicates of each of the toxicant series.

3.4.8.2 Mysidopsis. Determine the temperature, DO, and pH of the test solutions before the nauplii are added and at the 24- and 48hour exposure interval. Measure DO and pH in only one of the replicates of each of the toxicant series.

3.4.9 Testing laboratory. An ordinary heated or air-conditioned laboratory room with thermostatic controls suitable for maintaining the prescribed test temperatures generally will suffice to conduct the toxicity tests. Where ambient temperatures cannot

be controlled to 25±1 °C, use water baths with the necessary temperature controls.

3.4.10 Test containers. For tests with fish or mysids, use 1-liter glass beakers measuring approximately 10 cm in diameter. In conducting the test, add to each beaker 1 liter of the test solution or seawater formulation aerated to saturation with DO. To add the liter volume easily and accurately, use a large volume (1-liter) graduated cylinder. Process all required glassware before each test. Immerse in normal hexane for 10 minutes. Follow this with a thorough rinse with hot tap water; three hot detergent scrubs; an additional hot tap-water rinse; and three rinses with distilled water. Oven or air dry the glassware in a reasonably dust-free atmosphere.

3.5 Preparation of test concentrations. 3.5.1 Menidia. Place test jars (approximately 22.5 cm in height, 15 cm in diameter, 11 cm in diameter at the mouth) containing 2 liters of synthetic seawater on a reciprocal shaker. The shaker platform should be adapted to hold firmly six of the toxicity test jars. Add the desired amount of the petroleum product (if applicable) under test directly to each test jar. Dispense the appropriate amount of toxicant (if applicable) into the jars with a pipette. Tightly cap the test jars and shake for 5 minutes at approximately 315 to 333 2cm (0.75-inch) strokes per minute in a reciprocal shaker or at approximately 150 to 160 rpm on orbital shakers. At the completion of shaking, remove the jars from the shaker and dispense liter of the mixture to each of the 1-liter glass beakers. Randomly place beakers in a constant-temperature water bath or room, take water quality measurements, add fish, and initiate aeration.

3.5.2 Mysidopsis. 3.5.2.1 To prepare test solutions for products and oil/product mixtures, blend or mix the test solutions with an electric blender having: speeds of 10,000 rpm or less; a stainless-steel cutting assembly; and a 1-liter borosilicate jar. To minimize foaming, blend at speeds below 10,000 rpm.

3.5.2.2 For the product test solution, add 550 ml of the synthetic seawater to the jar, then with the use of a gas-tight calibrated glass syringe with a Teflon-tipped plunger, add 0.55 ml of the product and mix for 5 seconds.

3.5.2.3 For the oil test solution, add 550 ml of the synthetic seawater to the jar. Then with the use of a gas-tight calibrated glass syringe equipped with a Teflon-tipped plunger, add 0.55 ml of the oil and mix for 5 seconds.

3.5.2.4 For the oil/product mixture, add 550 ml of the synthetic seawater to the mixing jar. While the blender is in operation, add 0.5 ml of the oil under study with the use of a calibrated syringe with a Teflon-tipper plunger and then 0.05 ml of the product as indicated above. Blend for 5 seconds after addition of product. These additions provide test

solutions of the product, oil, and the oil/ product mixture at concentrations of 1,000 ppm.

3.5.2.5 Immediately after the test solutions are prepared, draw up the necessary amount of test solution with a gas-tight Teflon-tipped glass syringe of appropriate size and dispense into each of the five containers in each series. If the series of five concentrations to be tested are 10, 18, 32, 56, and 100 ppm, the amount of the test solution in the order of the concentrations listed above would be as follows: 10, 18, 32, 56, and 100 ml.

3.5.2.6 Each time a syringe is to be filled for dispensing to the series of test containers, start the mixer and withdraw the desired amount in the appropriate syringe while the mixer is in operation. Turn off immediately after the sample is taken to limit the loss of volatiles.

3.5.2.7 Use exploratory tests before the full-scale test is set up to determine the concentration of toxicant to be used in each of the five different concentrations. After adding the required amounts of liquid, bring the volume in each of the test containers up to 800 ml with the artificial seawater. To ensure keeping each of the series separate, designate on the lid of each container the date, the material under test, and its concentration.

3.5.2.8 When the desired concentrations are prepared, gently release into each beaker the 10 test Mysidopsis (previously transferred into 200 ml of medium). This provides a volume of 1 liter in each test chamber. A pair of standard cover glass forceps with flat, bent ends is an ideal tool for handling and tipping the small beaker without risk of contaminating the medium.

3.5.2.9 After adding the test animals, incubate the test beakers at 25±1 °C for 48 hours. Recommended lighting is 2,000 lumens/m2 (200 ft-c) of diffused, constant, fluorescent illumination.

3.5.2.10 Wash the blender thoroughly after use and repeat the above procedures for each series of tests. Wash the blender as follows: rinse with normal hexane; pour a strong solution of laboratory detergent into the blender to cover the blades; fill the container to about half of its volume with hot tap water; operate the blender for about 30 seconds at high speed; remove and rinse twice with hot tap water, mixing each rinse for 5 seconds at high speed; and then rinse twice with distilled water, mixing each rinse for 5 seconds at high speed.

3.6 Calculating and reporting. At the end of the test period, the toxicity tests are terminated and the LCso values are determined.

3.6.1 Calculations. The LCso is the concentration lethal to 50% of the test population. It can be calculated as an interpolated value based on percentages of organisms surviving at two or more concentrations, at which less than half and more than