an adequate set of test concentrations for a definitive test.

(4) The EPA Environmental Research Laboratory in Gulf Breeze, Florida prepared a Research and Development Report entitled Acute Toxicity of Eight Drilling Fluids to Mysid Shrimp (Mysidopsis bahia), May 1984 EPA-600/3-84-067. The Gulf Breeze data for drilling fluid number 1 are displayed in Table 1 for purposes of an example of the probit analysis described above. The SAS Probit Procedure (SAS Institute, Statistical Analysis System, Cary, North Carolina, 1982) was used to analyze these data. The 96-hour LC50 adjusted for the estimated spontaneous mortality rate is 3.3 percent SPP with 95 percent limits of 3.0 and 3.5 percent SPP with the 1 to 9 dilution. The estimated spontaneous mortality rate based on all of the data is 9.6 percent.

VI. References 1. Borthwick, Patrick W. 1978. Methods for acute static toxicity tests with mysid shrimp (Mysidopsis bahia). Bioassay Procedures for the Ocean Disposal Permit Program, (EPA-600 9–78-010: ) March.

2. Nimmo, D.R., T.L. Hamaker, and C.A. Somers.

1978. Culturing the mysid (Mysidopsis bahia) in flowing seawater or a static system. Bioassay Procedures for the Ocean Disposal Permit Program, [EPA-600/9 78–010): March

3. American Public Health Association et al. 1980. Standard Methods for the Examination of Water and Wastewater. Washington, DC, 15th Edition: 90-99.

4. U.S. Environmental Protection Agency, September 1991. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. EPA/600/4-90/027. Washington, DC, 4th Edition.

5. Finney, D.J. Probit Analysis. Cambridge University Press; 1971.

6. U.S. Environmental Protection Agency, May 1984. Acute Toxicity of Eight Drilling Fluids to Mysid Shrimp (Mysidopsis bahia). EPA-600/3–84-067.

V-C. The Partial Toricity Test for Evaluation of

Test Material (1) A partial test conducted according to EPA protocol can be used economically to demonstrate that a test material passes the toxicity test. The partial test cannot be used to estimate the LC-50 adjusted for natural response.

(2) To conduct a partial test follow the test protocol for preparation of the test material and organisms. Prepare the control (zero concentration), one test concentration (3 percent suspended particulate phase) and the reference toxicant according to the methods of the full test. A range finding test is not used for the partial test.

(3) Sixty test organisms are used for each test concentration. Find the number of test organisms killed in the control (zero percent SPP) concentration in the column labeled Xo of Table 2. If the number of organisms in the control (zero percent SPP) exceeds the table values, then the test is unacceptable and must be repeated. If the number of organisms killed in the 3 percent test concentration is less than or equal to corresponding number in the column labeled X, then the test material passes the partial toxicity test.

APPENDIX 3 TO SUBPART A OF PART

435-PROCEDURE FOR MIXING BASE

FLUIDS WITH SEDIMENTS This procedure describes a method for amending uncontaminated and nontoxic (control) sediments with the base fluids that are used to formulate synthetic-based drilling fluids and other non-aqueous drilling fluids. Initially, control sediments shall be press-sieved through a 2000 micron mesh sieve to remove large debris. Then presssieve the sediment through a 500 micron sieve to remove indigenous organisms that may prey on the test species or otherwise confound test results. Homogenize control sediment to limit the effects of settling that may have occurred during storage. Sediments should be homogenized before density determinations and addition of base fluid to control sediment. Because base fluids are

208-169 D-11

strongly hydrophobic and do not readily mix with sediment, care must be taken to ensure base fluids are thoroughly homogenized within the sediment. All concentrations are weight-to-weight (mg of base fluid to kg of dry control sediment). Sediment and base fluid mixing shall be accomplished by using the following method.

1. Determine the wet to dry ratio for the control sediment by weighing approximately 10 g subsamples of the screened and homogenized wet sediment into tared aluminum weigh pans. Dry sediment at 105 °C for 18–24 h. Remove sediment and cool in a desiccator until a constant weight is achieved. Reweigh the samples to determine the dry weight. Determine the wet/dry ratio by dividing the net wet weight by the net dry weight: [Wet Sediment Weight (g)]/[Dry Sediment

Weight (g)] = Wet to Dry Ratio [1]

2. Determine the density (g/mL) of the wet control or dilution sediment. This shall be used to determine total volume of wet sediment needed for the various test treatments. [Mean Wet Sediment Weight (g)]/[Mean Wet

Sediment Volume (mL)] = Wet Sediment Density (g/mL) [2]

3. To determine the amount of base fluid needed to obtain a test concentration of 500 mg base fluid per kg dry sediment use the following formulas:

Determine the amount of wet sediment required: [Wet Sediment Density (g/mL)] [Volume of Sediment Required per

Concentration (mL)] = Weight Wet Sediment Required per Conc. (g) [3]

Determine the amount of dry sediment in kilograms (kg) required for each concentration: {[Wet Sediment per concentration (g)]/[Mean

Wet to Dry Ratio}} * (1kg/1000g) = Dry Weight Sediment (kg) [4]

Finally, determine the amount of base fluid required to spike the control sediment at each concentration: (Conc. Desired (mg/kg)] * [Dry Weight Sedi

ment (kg)] = Base Fluid Required (mg) [5]

For spiking test substances other than pure base fluids (e.g., whole mud formulations), determine the spike amount as follows: (Conc. Desired (mL/kg)] * [Dry Weight Sedi

ment (kg)] x [Test Substance Density (g mL)] = Test Substance Required (g) [6]

4. For primary mixing, place appropriate amounts of weighed base fluid into stainless mixing bowls, tare the vessel weight, then add sediment and mix with a high-shear dispersing impeller for 9 minutes. The concentration of base fluid in sediment from this mix, rather than the nominal concentration, shall be used in calculating LCs. values.

5. Tests for homogeneity of base fluid in sediment are to be performed during the procedure development phase. Because of difficulty of homogeneously mixing base fluid with sediment, it is important to demonstrate that the base fluid is evenly mixed with sediment. The sediment shall be analyzed for total petroleum hydrocarbons (TPH) using EPA Methods 3550A and 8015M, with samples taken both prior to and after distribution to replicate test containers. Base-fluid content is measured as TPH. After mixing the sediment, a minimum of three replicate sediment samples shall be taken prior to distribution into test containers. After the test sediment is distributed to test containers, an additional three sediment samples shall be taken from three test containers to ensure proper distribution of base fluid within test containers. Base-fluid content results shall be reported within 48 hours of mixing. The coefficient of variation (CV) for the replicate samples must be less than 20%. If base-fluid content results are not within the 20% CV limit, the test sediment shall be remixed. Tests shall not begin until the CV is determined to be below the maximum limit of 20%. During the test, a minimum of three replicate containers shall be sampled to determine base-fluid content during each sampling period.

6. Mix enough sediment in this way to allow for its use in the preparation of all test concentrations and as a negative control. When commencing the sediment toxicity test, range-finding tests may be required to determine the concentrations that produce a toxic effect if these data are otherwise unavailable. The definitive test shall bracket the LCs., which is the desired endpoint. The results for the base fluids shall be reported in mg of base fluid per kg of dry sediment.

REFERENCES

American Society for Testing and Materials (ASTM). 1996. Standard Guide for Collection, Storage, Characterization, and Manipulation of Sediments for Toxicological Testing. ASTM E 1391–94. Annual Book of ASTM Standards, Volume 11.05, pp. 805-825.

Ditsworth, G.R., D.W. Schults and J.K.P. Jones. 1990. Preparation of benthic substrates for sediment toxicity testing, Environ. Toxicol. Chem. 9:1523-1529.

Suedel, B.C., J.H. Rodgers, Jr. and P.A. Clifford. 1993. Bioavailability of fluoranthene in freshwater sediment toxicity tests. Environ. Toxicol. Chem. 12:155-165.

U.S. EPA. 1994. Methods for Assessing the Toxicity of Sediment-associated Contaminants with Estuarine and Marine Amphipods. EPA/600/R-94/025. Office of Research and Development, Washington, DC. [66 FR 6901, Jan. 22, 2001)

APPENDIX 4 TO SUBPART A OF PART

435—DETERMINATION OF BIO-
DEGRADATION OF SYNTHETIC BASE
FLUIDS IN A MARINE CLOSED BOTTLE
TEST SYSTEM: SUMMARY OF MODI-
FICATIONS TO ISO 11734:1995

APPENDIX 5 TO SUBPART A OF PART

435—DETERMINATION OF CRUDE OIL
CONTAMINATION IN NON-AQUEOUS
DRILLING FLUIDS BY GAS CHROMA-
TOGRAPHY/

MASS SPECTROMETRY (GC/MS)

1.0 SCOPE AND APPLICATION

The six modifications specified in this Appendix shall apply to the determination of the biodegradability of synthetic base fluids as measured by ISO 11734:1995. These modifications make the test more applicable to a marine environment and are listed below:

1. The laboratory shall use sea water in place of freshwater media.

1.1 The sea water may be either natural or synthetic. The allowable salinity range is 20-30 ppt.

1.2 To reduce the shock to the microorganisms in the sediment, the salinity of the sediment's porewater shall be between 20-30 ppt.

2. The laboratory shall use natural marine or estuarine sediments in place of digested sludge as an inoculum. The VS of the sediments must be no less than 2%.

2.1 Sediment should be used for testing as soon as possible after field collection. If required, the laboratory can store the sediment for a maximum period of two months prior to use. The test sediment shall be stored in the dark at 4 °C.

2.2 The laboratory shall use the sediment mixing procedure specified in Appendix 3 to Subpart A of part 435 to spike the test sediment with base fluids. The final concentration will be 2000 mg carbon Kg dry weight sediment. No less than 25 g dry weight of the spiked sediment shall be used per 125 ml serum bottle. The volume of sediment and seawater in the bottle shall be 75 ml.

3. The temperature of incubation shall be 29 +1 °C.

4. The pH is maintained at the level of natural sea water, not at 7.0 as referenced in ISO 11734:1995.

5. The optional use of a trace metals solution as specified in method ISO 11734:1995 shall not be used as part of these test modifications.

6. The laboratory shall conduct the test for 275 days. The laboratory may seek approval of alternate test durations under the approval procedures specified at 40 CFR 136.4 and 136.5. Any modification of this method, beyond those expressly permitted, shall be considered a major modification subject to application and approval of alternate test procedures under 40 CFR 136.4 and 136.5. (66 FR 6901, Jan. 22, 2001)

1.1 This method determines crude (formation) oil contamination, or other petroleum oil contamination, in non-aqueous drilling fluids (NAFs) by comparing the gas chromatography/mass spectrometry (GC/MS) fingerprint scan and extracted ion scans of the test sample to that of an uncontaminated sample.

1.2 This method can be used for monitoring oil contamination of NAFs or monitoring oil contamination of the base fluid used in the NAF formulations.

1.3 Any modification of this method beyond those expressly permitted shall be considered as a major modification subject to application and approval of alternative test procedures under 40 CFR 136.4 and 136.5.

1.4 The gas chromatography/mass spectrometry portions of this method are restricted to use by, or under the supervision of analysts experienced in the use of GC/MS and in the interpretation of

gas chromatograms and extracted ion scans. Each laboratory that uses this method must generate acceptable results using the procedures described in Sections 7, 9.2, and 12 of this appendix.

2.0 SUMMARY OF METHOD 2.1 Analysis of NAF for crude oil contamination is a step-wise process. The analyst first performs a qualitative assessment of the presence or absence of crude oil in the sample. If crude oil is detected during this qualitative assessment, the analyst must perform a quantitative analysis of the crude oil concentration.

2.2 A sample of NAF is centrifuged to obtain a solids free supernate.

2.3 The test sample is prepared by removing an aliquot of the solids free supernate, spiking it with internal standard, and analyzing it using GC/MS techniques. The components are separated by the gas chromatograph and detected by the mass spectrometer.

2.4 Qualitative identification of crude oil contamination is performed by comparing the Total Ion Chromatograph (TIC) scans and Extracted Ion Profile (EIP) scans of test sample to that of uncontaminated base fluids, and examining the profiles for chromatographic signatures diagnostic of oil contamination.

2.5 The presence or absence of crude oil contamination observed in the full scan profiles and selected extracted ion profiles determines further sample quantitation and reporting requirements.

2.6 If crude oil is detected in the qualitative analysis, quantitative analysis must be performed by calibrating the GC/MS using a designated NAF spiked with known concentrations of a designated oil.

2.7 Quality is assured through reproducible calibration and testing of GC/

MS system and through analysis of quality control samples.

5.2 Unknown samples may contain high concentration of volatile toxic compounds. Sample containers should be opened in a hood and handled with gloves to prevent exposure. In addition, all sample preparation should be conducted in a fume hood to limit the potential exposure to harmful contaminates.

5.3 This method does not address all safety issues associated with its use. The laboratory is responsible for maintaining a safe work environment and a current awareness file of OSHA regulations regarding the safe handling of the chemicals specified in this method. A reference file of material safety data sheets (MSDSS) shall be available to all personnel involved in these analyses. Additional references to laboratory safety can be found in References 16.1 through 16.3.

5.4 NAF base fluids may cause skin irritation, protective gloves are recommended while handling these samples.

3.0 DEFINITIONS 3.1 A NAF is one in which the continuous-phase is a water immiscible fluid such as an oleaginous material (e.g., mineral oil, enhance mineral oil, paraffinic oil, or synthetic material such as olefins and vegetable esters).

3.2 TIC—Total Ion Chromatograph. 3.3 EIP-Extracted Ion Profile.

3.4 TCB-1,3,5-trichlorobenzene is used as the internal standard in this method.

3.5 SPTM-System Performance Test Mix standards are used to establish retention times and monitor detection levels.

4.0 INTERFERENCES AND LIMITATIONS 4.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or elevated baselines causing misinterpretation of chromatograms.

4.2 All Materials used in the analysis shall be demonstrated to be free from interferences by running method blanks. Specific selection of reagents and purification of solvents by distillation in all-glass systems may be required.

4.3 Glassware shall be cleaned by rinsing with solvent and baking at 400 °C for a minimum of 1 hour.

4.4 Interferences may vary from source to source, depending on the diversity of the samples being tested.

4.5 Variations in and additions of base fluids and/or drilling fluid additives (emulsifiers, dispersants, fluid loss control agents, etc.) might also cause interferences and misinterpretation of chromatograms.

4.6 Difference in light crude oils, medium crude oils, and heavy crude oils will result in different responses and thus different interpretation of scans and calculated percentages.

6.0 APPARATUS AND MATERIALS NOTE: Brand names, suppliers, and part numbers are for illustrative purposes only. No endorsement is implied. Equivalent performance may be achieved using apparatus and materials other than those specified here, but demonstration of equivalent performance meeting the requirements of this method is the responsibility of the laboratory.

6.1 Equipment for glassware cleaning.

6.1.1 Laboratory sink with overhead fume hood.

6.1.2 Kiln-Capable of reaching 450 °C within 2 hours and holding 450 °C within +10 °C, with temperature controller and safety switch (Cress Manufacturing Co., Santa Fe Springs, CA B31H or X31TS or equivalent).

6.2 Equipment for sample preparation. 6.2.1 Laboratory fume hood.

6.2.2 Analytical balance-Capable of weighing 0.1 mg.

6.2.3 Glassware.

6.2.3.1 Disposable pipettes—Pasteur, 150 mm long by 5 mm ID (Fisher Scientific 13678-6A, or equivalent) baked at 400 °C for a minimum of 1 hour.

6.2.3.2 Glass volumetric pipettes or gas tight syringes--1.0-mL 1% and 0.5-mL +1%.

6.2.3.3 Volumetric flasks-Glass, class A, 10-mL, 50-mL and 100-ML.

6.2.3.4Sample vials—Glass, 1- to 3-mL (baked at 400 °C for a minimum of 1 hour) with PTFE-lined screw or crimp cap.

6.2.3.5 Centrifuge and centrifuge tubesCentrifuge capable of 10,000 rpm, or better, (International Equipment Co., IEC Centra MP4 or equivalent) and 50-ml centrifuge tubes (Nalgene, Ultratube, Thin Wall 25x89 mm, #3410–2539).

6.3 Gas Chromatograph/Mass Spectrometer (GC/MS):

5.0 SAFETY 5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely determined; however each chemical shall be treated as a potential health hazard. Exposure to these chemicals should be reduced to the lowest possible level.

6.3.1 Gas Chromatograph-An analytical system complete with a temperature-programmable gas chromatograph suitable for splitsplitiess injection and all required accessories, including syringes, analytical columns, and gases.

6.3.1.1 Column 30 m (or 60 m) x 0.32 mm ID (or 0.25 mm ID) 1 um film thickness (or 0.25 um film thickness) silicone-coated fusedsilica capillary column (J&W Scientific DB5 or equivalent).

6.3.2 Mass Spectrometer--Capable of scanning from 35 to 500 amu every 1 sec or less, using 70 volts (nominal) electron energy in the electron impact ionization mode (Hewlett Packard 5970MS or comparable).

6.3.3 GC/MS interface-the interface is a capillary-direct interface from the GC to the MS.

6.3.4Data System--A computer system must be interfaced to the mass spectrometer. The system must allow the continuous acquisition and storage on machine-readable media of all mass spectra obtained throughout the duration of the chromatographic program. The computer must have software that can search any GC/MS data file for ions of a specific mass and that can plot such ion abundance versus retention time or scan number. This type of plot is defined as an Extracted Ion Current Profile (EIP). Software must also be available that allows integrating the abundance in any total ion chromatogram (TIC) or EIP between specified retention time or scan-number limits. It is advisable that the most recent version of the EPA/NIST Mass Spectral Library be available.

7.0 REAGENTS AND STANDARDS 7.1 Methylene chloride-Pesticide grade or equivalent. Use when necessary for sample dilution.

7.2 Standards-Prepare from pure individual standard materials or purchase as certified solutions. If compound purity is 96% or greater, the weight may be used without correction to compute the concentration of the standard.

7.2.1 Crude Oil Reference Obtain a sample of a crude oil with a known API gravity. This oil shall be used in the calibration procedures.

7.2.2 Synthetic Base Fluid-Obtain a sample of clean internal olefin (10) Lab drilling fluid (as sent from the supplier-has not been circulated downhole). This drilling fluid shall be used in the calibration procedures.

7.2.3 Internal standard-Prepare a 0.01 g mL solution of 1,3,5-trichlorobenzene (TCB). Dissolve 1.0 g of TCB in methylene chloride and dilute to volume in a 100-ml volumetric flask. Stopper, vortex, and transfer the solution to a 150-ml bottle with PTFE-lined cap. Label appropriately, and store at -5 °C to 20 °C. Mark the level of the meniscus on the bottle to detect solvent loss.

7.2.4 GC/MS system performance test mix (SPTM) standards—The SPTM standards shall contain octane, decane, dodecane, tetradecane, tetradecene, toluene, ethylbenzene, 1,2,4-trimethylbenzene, 1methylnaphthalene

and

1,3dimethylnaphthalene. These compounds can be purchased individually or obtained as a mixture (i.e., Supelco, Catalog No. 4-7300). Prepare a high concentration of the SPTM standard at 62.5 mg/ml in methylene chloride. Prepare a medium concentration SPTM standard at 1.25 mg/mL by transferring 1.0 mL of the 62.5 mg/mL solution into a 50 mL volumetric flask and diluting to the mark with methylene chloride. Finally, prepare a low concentration SPTM standard at 0.125 mg/mL by transferring 1.0 mL of the 1.25 mg/ mL solution into a 10-ml volumetric flask and diluting to the mark with methylene chloride.

7.2.5 Crude oil/drilling fluid calibration standards--Prepare a 4-point crude oil/drilling fluid calibration at concentrations of 0% (no spike-clean drilling fluid), 0.5%, 1.0%, and 2.0% by weight according to the procedures outlined in this appendix using the Reference Crude Oil:

7.2.5.1 Label 4 jars with the following identification: Jar 1–0% Ref-IOLab, Jar 20.5%Ref-IOLab, Jar 3-1%Ref-IOLab, and Jar 42%Ref-IOLab.

7.2.5.2 Weigh 4, 50-g aliquots of well mixed IO Lab drilling fluid into each of the 4 jars.

7.2.5.3 Add Reference Oil at 0.5%, 1.0%, and 2.0% by weight to jars 2, 3, and 4 respectively. Jar 1 shall not be spiked with Reference Oil in order to retain a “0%" oil concentration.

7.2.5.4 Thoroughly mix the contents of each of the 4 jars, using clean glass stirring rods.

7.2.5.5 Transfer (weigh) a 30-g aliquot from Jar 1 to a labeled centrifuge tube. Centrifuge the aliquot for a minimum of 15 min at approximately 15,000 rpm, in order to obtain a solids free supernate. Weigh 0.5 g of the supernate directly into a tared and appropriately labeled GC straight vial. Spike the 0.5-g supernate with 500 uL of the 0.01g/mL 1,3,5-trichlorobenzene internal standard solution (see Section 7.2.3 of this appendix), cap with a Teflon lined crimp cap, and vortex for ca. 10 sec.

7.2.5.6 Repeat step 7.2.5.5 except use an aliquot from Jar 2.

7.2.5.7 Repeat step 7.2.5.5 except use an aliquot from Jar 3.

7.2.5.8 Repeat step 7.2.5.5 except use an aliquot from Jar 4.

7.2.5.9 These 4 crude oil drilling fluid calibration standards are now used for qualitative and quantitative GC/MS analysis.

7.2.6 Precision and recovery standard (mid level crude oil/drilling fluid calibration standard)Prepare a mid point crude oil/