DRAFT FOR PUBLIC COMMENT

Analytic Methods for the Oil and Gas Extraction Point Source Category

U.S. Environmental Protection Agency

Engineering and Analysis Division

Office of Water

1200 Pennsylvania Avenue, NW

Washington, D.C. 20460

September 2009 [This date will be update for final document.]

EPA-821-R-09-013

CONTENTS

Page

  TOC \o "1-2" \h \z \u    HYPERLINK \l "_Toc249333060"  1.	Summary of
Methods	  PAGEREF _Toc249333060 \h  1-1

  HYPERLINK \l "_Toc249333061"  1.1	Static Sheen Test (EPA Method 1617)
 PAGEREF _Toc249333061 \h  1-5

  HYPERLINK \l "_Toc249333062"  1.2	Drilling Fluids Toxicity Test (EPA
Method 1619)	  PAGEREF _Toc249333062 \h  1-5

  HYPERLINK \l "_Toc249333063"  1.3	Procedure for Mixing Base Fluids
with Sediments (EPA Method 1646)	  PAGEREF _Toc249333063 \h  1-5

  HYPERLINK \l "_Toc249333064"  1.4	Protocol for the Determination of
Degradation of Non Aqueous Base Fluids in a Marine Closed Bottle
Biodegradation Test System: Modified ISO 11734:1995 (EPA Method 1647)
PAGEREF _Toc249333064 \h  1-6

  HYPERLINK \l "_Toc249333065"  1.5	Determination of Crude Oil
Contamination in Non-Aqueous Drilling Fluids by Gas Chromatography/Mass
Spectrometry (GC/MS) (EPA Method 1655)	  PAGEREF _Toc249333065 \h  1-7

  HYPERLINK \l "_Toc249333066"  1.6	Reverse Phase Extraction (RPE)
Method for Detection of Oil Contamination in Non-Aqueous Drilling Fluids
(NAF) (EPA Method 1670)	  PAGEREF _Toc249333066 \h  1-7

  HYPERLINK \l "_Toc249333067"  1.7	Determination of the Amount Of
Non-Aqueous Drilling Fluid (NAF) Base Fluid from Drill Cuttings by a
Retort Chamber (Derived From API Recommended Practice 13B–2) (EPA
Method 1674)	  PAGEREF _Toc249333067 \h  1-8

  HYPERLINK \l "_Toc249333068"  1.8	PAH Content of Oil by HPLC/UV (EPA
Method 1654, Revision A)	  PAGEREF _Toc249333068 \h  1-8

  HYPERLINK \l "_Toc249333069"  1.9	Previous Publication of Oil and Gas
Extraction Point Source Category Analytic Methods	  PAGEREF
_Toc249333069 \h  1-8

  HYPERLINK \l "_Toc249333070"  2.	Static Sheen Test (EPA Method 1617)
PAGEREF _Toc249333070 \h  2-10

  HYPERLINK \l "_Toc249333071"  2.1	Scope and Application	  PAGEREF
_Toc249333071 \h  2-10

  HYPERLINK \l "_Toc249333072"  2.2	Summary of Method	  PAGEREF
_Toc249333072 \h  2-10

  HYPERLINK \l "_Toc249333073"  2.3	Interferences	  PAGEREF
_Toc249333073 \h  2-10

  HYPERLINK \l "_Toc249333074"  2.4	Apparatus, Materials, and Reagents
PAGEREF _Toc249333074 \h  2-10

  HYPERLINK \l "_Toc249333075"  2.5	Calibration	  PAGEREF _Toc249333075
\h  2-11

  HYPERLINK \l "_Toc249333076"  2.6	Quality Control Procedures	  PAGEREF
_Toc249333076 \h  2-11

  HYPERLINK \l "_Toc249333077"  2.7	Sample Collection and Handling
PAGEREF _Toc249333077 \h  2-11

  HYPERLINK \l "_Toc249333078"  2.8	Procedure	  PAGEREF _Toc249333078 \h
 2-12

  HYPERLINK \l "_Toc249333079"  3.	Drilling Fluids Toxicity Test (EPA
Method 1619)	  PAGEREF _Toc249333079 \h  3-1

  HYPERLINK \l "_Toc249333080"  3.1	Scope and Application	  PAGEREF
_Toc249333080 \h  3-1

  HYPERLINK \l "_Toc249333081"  3.2	Summary of Method	  PAGEREF
_Toc249333081 \h  3-1

  HYPERLINK \l "_Toc249333082"  3.3	Sample Collection	  PAGEREF
_Toc249333082 \h  3-1

  HYPERLINK \l "_Toc249333083"  3.4	Suspended Particulate Phase Sample
Preparation	  PAGEREF _Toc249333083 \h  3-2

  HYPERLINK \l "_Toc249333084"  3.5	Guidance for Performing Suspended
Particulate Phase Toxicity Tests Using Mysidopsis bahia	  PAGEREF
_Toc249333084 \h  3-4

  HYPERLINK \l "_Toc249333085"  3.6	Methods for Positive Control Tests
(Reference Toxicant)	  PAGEREF _Toc249333085 \h  3-6

  HYPERLINK \l "_Toc249333086"  3.7	Randomization Procedure	  PAGEREF
_Toc249333086 \h  3-7

  HYPERLINK \l "_Toc249333087"  3.8	References	  PAGEREF _Toc249333087
\h  3-12

  HYPERLINK \l "_Toc249333088"  4.	Procedure for Mixing Base Fluids With
Sediments (EPA Method 1646)	  PAGEREF _Toc249333088 \h  4-1

  HYPERLINK \l "_Toc249333089"  4.1	Determining the Wet to Dry Ratio for
the Control Sediment	  PAGEREF _Toc249333089 \h  4-1

  HYPERLINK \l "_Toc249333090"  4.2	Determining the Density of the Wet
Control or Dilution Sediment	  PAGEREF _Toc249333090 \h  4-1

  HYPERLINK \l "_Toc249333091"  4.3	Determining the Amount of Base Fluid
Needed	  PAGEREF _Toc249333091 \h  4-1

  HYPERLINK \l "_Toc249333092"  4.4	Primary Mixing	  PAGEREF
_Toc249333092 \h  4-2

  HYPERLINK \l "_Toc249333093"  4.5	Testing for Homogeneity of Base
Fluid	  PAGEREF _Toc249333093 \h  4-2

  HYPERLINK \l "_Toc249333094"  4.6	Commencing the Sediment Toxicity
Test	  PAGEREF _Toc249333094 \h  4-2

  HYPERLINK \l "_Toc249333095"  4.7	References	  PAGEREF _Toc249333095
\h  4-3

  HYPERLINK \l "_Toc249333096"  5.	Protocol for the Determination of
Degradation of Non-Aqueous Base Fluids in a Marine Closed Bottle
Biodegradation Test System: Modified ISO 11734:1995 (EPA Method 1647)
PAGEREF _Toc249333096 \h  5-1

  HYPERLINK \l "_Toc249333097"  5.1	Summary of Method	  PAGEREF
_Toc249333097 \h  5-1

  HYPERLINK \l "_Toc249333098"  5.2	System Requirements	  PAGEREF
_Toc249333098 \h  5-1

  HYPERLINK \l "_Toc249333099"  5.3	Test Set Up	  PAGEREF _Toc249333099
\h  5-2

  HYPERLINK \l "_Toc249333100"  5.4	Concentration Verification Chemical
Analyses	  PAGEREF _Toc249333100 \h  5-6

  HYPERLINK \l "_Toc249333101"  5.5	Gas Monitoring Procedures	  PAGEREF
_Toc249333101 \h  5-7

  HYPERLINK \l "_Toc249333102"  5.6	Test Acceptability and
Interpretation	  PAGEREF _Toc249333102 \h  5-8

  HYPERLINK \l "_Toc249333103"  5.7	Methane Measurement	  PAGEREF
_Toc249333103 \h  5-9

  HYPERLINK \l "_Toc249333104"  5.8	Concentration Verification Analysis
 PAGEREF _Toc249333104 \h  5-11

  HYPERLINK \l "_Toc249333105"  5.9	Program Quality Assurance and
Quality Control	  PAGEREF _Toc249333105 \h  5-12

  HYPERLINK \l "_Toc249333106"  6.	Determination of Crude Oil
Contamination in Non-Aqueous Drilling Fluids by Gas Chromatography/Mass
Spectrometry (GC/MS) (EPA Method 1655)	  PAGEREF _Toc249333106 \h  6-1

  HYPERLINK \l "_Toc249333107"  6.1	Scope and Application	  PAGEREF
_Toc249333107 \h  6-1

  HYPERLINK \l "_Toc249333108"  6.2	Summary of Method	  PAGEREF
_Toc249333108 \h  6-1

  HYPERLINK \l "_Toc249333109"  6.3	Definitions	  PAGEREF _Toc249333109
\h  6-2

  HYPERLINK \l "_Toc249333110"  6.4	Interferences and Limitations
PAGEREF _Toc249333110 \h  6-2

  HYPERLINK \l "_Toc249333111"  6.5	Safety	  PAGEREF _Toc249333111 \h
6-2

  HYPERLINK \l "_Toc249333112"  6.6	Apparatus and Materials	  PAGEREF
_Toc249333112 \h  6-3

  HYPERLINK \l "_Toc249333113"  6.7	Reagents and Standards	  PAGEREF
_Toc249333113 \h  6-4

  HYPERLINK \l "_Toc249333114"  6.8	Sample Collection Preservation and
Storage	  PAGEREF _Toc249333114 \h  6-6

  HYPERLINK \l "_Toc249333115"  6.9	Quality Control	  PAGEREF
_Toc249333115 \h  6-6

  HYPERLINK \l "_Toc249333116"  6.10	Calibration	  PAGEREF _Toc249333116
\h  6-9

  HYPERLINK \l "_Toc249333117"  6.11	Procedure	  PAGEREF _Toc249333117
\h  6-11

  HYPERLINK \l "_Toc249333118"  6.12	Calculations	  PAGEREF
_Toc249333118 \h  6-15

  HYPERLINK \l "_Toc249333119"  6.13	Method Performance	  PAGEREF
_Toc249333119 \h  6-16

  HYPERLINK \l "_Toc249333120"  6.14	Pollution Prevention	  PAGEREF
_Toc249333120 \h  6-16

  HYPERLINK \l "_Toc249333121"  6.15	Waste Management	  PAGEREF
_Toc249333121 \h  6-16

  HYPERLINK \l "_Toc249333122"  6.16	References	  PAGEREF _Toc249333122
\h  6-17

  HYPERLINK \l "_Toc249333123"  6.17	Schematic Flowchart for Qualitative
Identification	  PAGEREF _Toc249333123 \h  6-18

  HYPERLINK \l "_Toc249333124"  7.	Reverse Phase Extraction (RPE) Method
for Detection of Oil Contamination in Non-Aqueous Drilling Fluids (NAF)
(EPA Method 1670)	  PAGEREF _Toc249333124 \h  7-1

  HYPERLINK \l "_Toc249333125"  7.1	Scope and Application	  PAGEREF
_Toc249333125 \h  7-1

  HYPERLINK \l "_Toc249333126"  7.2	Summary of Method	  PAGEREF
_Toc249333126 \h  7-1

  HYPERLINK \l "_Toc249333127"  7.3	Definitions	  PAGEREF _Toc249333127
\h  7-1

  HYPERLINK \l "_Toc249333128"  7.4	Interferences	  PAGEREF
_Toc249333128 \h  7-2

  HYPERLINK \l "_Toc249333129"  7.5	Safety	  PAGEREF _Toc249333129 \h
7-2

  HYPERLINK \l "_Toc249333130"  7.6	Equipment and Supplies	  PAGEREF
_Toc249333130 \h  7-2

  HYPERLINK \l "_Toc249333131"  7.7	Reagents and Standards	  PAGEREF
_Toc249333131 \h  7-4

  HYPERLINK \l "_Toc249333132"  7.8	Sample Collection, Preservation, and
Storage	  PAGEREF _Toc249333132 \h  7-4

  HYPERLINK \l "_Toc249333133"  7.9	Quality Control	  PAGEREF
_Toc249333133 \h  7-4

  HYPERLINK \l "_Toc249333134"  7.10	Calibration and Standardization
PAGEREF _Toc249333134 \h  7-7

  HYPERLINK \l "_Toc249333135"  7.11	Procedure	  PAGEREF _Toc249333135
\h  7-7

  HYPERLINK \l "_Toc249333136"  7.12	Data Analysis and Calculations
PAGEREF _Toc249333136 \h  7-9

  HYPERLINK \l "_Toc249333137"  7.13	Method Performance	  PAGEREF
_Toc249333137 \h  7-9

  HYPERLINK \l "_Toc249333138"  7.14	Pollution Prevention	  PAGEREF
_Toc249333138 \h  7-9

  HYPERLINK \l "_Toc249333139"  7.15	Waste Management	  PAGEREF
_Toc249333139 \h  7-10

  HYPERLINK \l "_Toc249333140"  7.16	References	  PAGEREF _Toc249333140
\h  7-10

  HYPERLINK \l "_Toc249333141"  8.	Determination of the Amount of
Non-Aqueous Drilling Fluid (NAF) Base Fluid from Drill Cuttings by a
Retort Chamber (Derived from API Recommended Practice 13B–2) (EPA
Method 1674)	  PAGEREF _Toc249333141 \h  8-1

  HYPERLINK \l "_Toc249333142"  8.1	Description	  PAGEREF _Toc249333142
\h  8-1

  HYPERLINK \l "_Toc249333143"  8.2	Equipment	  PAGEREF _Toc249333143 \h
 8-1

  HYPERLINK \l "_Toc249333144"  8.3	Procedure	  PAGEREF _Toc249333144 \h
 8-2

  HYPERLINK \l "_Toc249333145"  8.4	Calculations	  PAGEREF _Toc249333145
\h  8-3

  HYPERLINK \l "_Toc249333146"  8.5	Requirements for Sampling Cuttings
Discharge Streams for use with this Method	  PAGEREF _Toc249333146 \h
8-9

  HYPERLINK \l "_Toc249333147"  8.6	Best Management Practices (BMPs) for
use with this Method	  PAGEREF _Toc249333147 \h  8-12

  HYPERLINK \l "_Toc249333148"  9.	PAH Content of Oil by HPLC/UV (EPA
Method 1654, Revision A)	  PAGEREF _Toc249333148 \h  9-1

  HYPERLINK \l "_Toc249333149"  9.1	Scope and Application	  PAGEREF
_Toc249333149 \h  9-1

  HYPERLINK \l "_Toc249333150"  9.2	Summary of Method	  PAGEREF
_Toc249333150 \h  9-1

  HYPERLINK \l "_Toc249333151"  9.3	Interferences	  PAGEREF
_Toc249333151 \h  9-1

  HYPERLINK \l "_Toc249333152"  9.4	Safety	  PAGEREF _Toc249333152 \h
9-2

  HYPERLINK \l "_Toc249333153"  9.5	Apparatus and Materials	  PAGEREF
_Toc249333153 \h  9-2

  HYPERLINK \l "_Toc249333154"  9.6	Reagents	  PAGEREF _Toc249333154 \h
9-4

  HYPERLINK \l "_Toc249333155"  9.7	Calibration	  PAGEREF _Toc249333155
\h  9-5

  HYPERLINK \l "_Toc249333156"  9.8	Quality Assurance/Quality Control
PAGEREF _Toc249333156 \h  9-6

  HYPERLINK \l "_Toc249333157"  9.9	Sample Collection, Preservation, and
Handling	  PAGEREF _Toc249333157 \h  9-9

  HYPERLINK \l "_Toc249333158"  9.10	Dilution of Oil and Extracts
PAGEREF _Toc249333158 \h  9-9

  HYPERLINK \l "_Toc249333159"  9.11	High-Performance Liquid
Chromatography	  PAGEREF _Toc249333159 \h  9-10

  HYPERLINK \l "_Toc249333160"  9.12	HPLC System and Laboratory
Performance	  PAGEREF _Toc249333160 \h  9-11

  HYPERLINK \l "_Toc249333161"  9.13	Qualitative Identification
PAGEREF _Toc249333161 \h  9-12

  HYPERLINK \l "_Toc249333162"  9.14	Quantitative Determination
PAGEREF _Toc249333162 \h  9-12

  HYPERLINK \l "_Toc249333163"  9.15	Method Performance	  PAGEREF
_Toc249333163 \h  9-13

  HYPERLINK \l "_Toc249333164"  9.16	References	  PAGEREF _Toc249333164
\h  9-13

LIST OF TABLES

Page

  TOC \t "Table Heading" \c  Table 1-1. Methods by Waste and Pollutant,
Subpart A − Offshore Subcategory	  PAGEREF _Toc249333165 \h  1-2

Table 1-2. Methods by Waste and Pollutant, Subpart D − Coastal
Subcategory	  PAGEREF _Toc249333166 \h  1-4

Table 1-3. EPA Method Numbers for Oil and Gas Extraction Point Source
Category Analytical Methods and Prior CFR References	  PAGEREF
_Toc249333167 \h  1-9

Table 3-1. Example of a Randomization Schedule	  PAGEREF _Toc249333168
\h  3-9

Table 3-2. Listing of Acute Toxicity Test Data (August 1983 to September
1983) With Eight Generic Drilling Fluids and Mysid Shrimp	  PAGEREF
_Toc249333169 \h  3-11

Table 3-3. Partial Toxicity Test Passing Criteria	  PAGEREF
_Toc249333170 \h  3-12

Table 5-1. Test Acceptability Criteria	  PAGEREF _Toc249333171 \h  5-9

Table 6-1. Gas Chromatograph/Mass Spectrometer (GC/MS) Operation
Conditions	  PAGEREF _Toc249333172 \h  6-9

Table 6-2. Approximate Retention Time for Compounds	  PAGEREF
_Toc249333173 \h  6-10

Table 6-3. Recommended Ion Mass Numbers	  PAGEREF _Toc249333174 \h  6-13

Table 9-1. Performance Data and Method Acceptance Criteria for PAH
PAGEREF _Toc249333175 \h  9-14

Table 9-2. HPLC Calibration Data	  PAGEREF _Toc249333176 \h  9-14

LIST OF FIGURES

Page

  TOC \t "Figure Title" \c  Figure 3-1. Mysid Randomization Procedure
PAGEREF _Toc249333177 \h  3-8

Figure 6-1.  Schematic Flowchart for Qualitative Identification
PAGEREF _Toc249333178 \h  6-18

Figure 9-1. Liquid Chromatography of the Three-Component Standard and of
No. 2 Diesel Oil	  PAGEREF _Toc249333179 \h  9-15

Summary of Methods

This document is a compilation of all but two analytical methods
necessary for demonstrating compliance with the requirements of 40 CFR
Part 435, effluent limitations guidelines and standards for the Oil and
Gas Extraction Point Source Category. These methods are tests for
regulated pollutant parameters. The waste sources and pollutant
parameters regulated in 40 CFR Part 435 are listed in the following
tables along with the required analytical or test method, and the
subpart that cites the method.   REF _Ref237924987 \h  Table 1-1
presents the waste sources and pollutant parameters regulated by Subpart
A – Offshore Subcategory.   REF _Ref239478693 \h  Table 1-2  presents
the same information for Subpart D – Coastal Subcategory.

These methods were developed during two rulemakings: Offshore
Subcategory (March 4, 1993; 58 FR 12454) and the Synthetic-based
Drilling Fluids (SBF) Effluent Guidelines (January 22, 2001; 66 FR
6849). These analytic methods were developed in consultation and
collaboration with industry. For example, the analytic methods for the
Offshore Subcategory were developed with industry in the 1980’s and
included the Static Sheen Test and Drilling Fluids Toxicity Test (see
August 26, 1985; 50 FR 34596). EPA again worked very closely with
industry and adopted their recommendations in promulgating additional
analytical methods for the Synthetic-based Drilling Fluids Effluent
Guidelines (see January 22, 2001; 66 FR 6893). These additional
analytical methods included tests for PAH content (as phenanthrene),
sediment toxicity, biodegradation rate, and percentage of basefluid on
drill cuttings (API retort method). Prior to the 2010 Methods Update
Rule (XXX – insert date and FR citation for final rule) EPA published
these methods in the Code of Federal Regulations. The 2010 Methods
Update Rule updates the Oil and Gas Extraction Point Source Category by
incorporating these methods into the effluent guidelines by referencing
this document.

As previously noted there are two methods that are not included in this
document. The first method not included in this document relates to the
sediment toxicity effluent guidelines: Standard Guide for Conducting
10-day Static Sediment Toxicity Tests with Marine and Estuarine
Amphipods (ASTM E 1367–92). EPA requires operators using this method
to use Leptocheirus plumulosus as the test organism and the sediment
preparation procedures specified in EPA Method 1646. EPA incorporated
this method by reference in accordance with 5 U.S.C. 552(a) and 1 CFR
part 51. Copies may be obtained from the American Society for Testing
and Materials, 100 Barr Harbor Drive, West Conshohocken, PA, 19428. A
copy may also be inspected at EPA's Water Docket, 1200 Pennsylvania
Ave., NW., Washington, DC 20460.

The second method not included in this document relates to Cook Inlet,
Alaska, operators that are regulated by the Coastal subcategory (Subpart
D). The Coastal subcategory prohibits the discharge non-aqueous drill
cuttings unless there are technical limitations. Appendix 1 of 40 CFR
Part 435 Subpart D provides the method for permit writers to determine
when operators qualify for an exemption from the discharge prohibition.
For those operators that quality for the exemption from the discharge
prohibition EPA requires that these operators meet the same stock
limitations and discharge limitations for drill cuttings associated with
non-aqueous drilling fluids for operators in Offshore waters (see 40 CFR
§435.13 and §435.15) in order to discharge drill cuttings associated
with non-aqueous drilling fluids. This method remains in 40 CFR 435 and
is not included in this document.

The remainder of this section provides a summary of the analytical
methods used for Part 435.

Table   STYLEREF 1 \s  1 -  SEQ Table \* ARABIC \s 1  1 . Methods by
Waste and Pollutant, Subpart A − Offshore Subcategory

Waste source	Regulated Pollutant Parameter	Analytical/Test Method	Cited
in 40 CFR Part

Produced Water	Oil & grease	40 CFR Part 136	§435.12, §435.13,
§435.14, §435.15

Water-based drilling fluids and associated drill cuttings	SPP Toxicity
Drilling Fluids Toxicity Test

EPA Method 1619	§435.13, §435.15

	Free oil	Static Sheen Test

EPA Method 1617	§435.12, §435.13, §435.14, §435.15

	Diesel oil †	ASTM specification D975-91	§435.13, §435.15

	Mercury	40 CFR Part 136	§435.13, §435.15

	Cadmium	40 CFR Part 136	§435.13, §435.15

Drill cuttings associated with non-aqueous drilling fluids: Discharge
limitations	Diesel oil †	ASTM specification D975-91	§435.13, §435.15

	Free oil	Static Sheen Test

EPA Method 1617	§435.12, §435.14

	SPP Toxicity	Drilling Fluids Toxicity Test

EPA Method 1619	§435.13, §435.15

	Drilling fluid sediment toxicity (4-Day Test)	Standard Guide for
Conducting 10-day Static Sediment Toxicity Tests with Marine and
Estuarine Amphipods ASTM E 1367–92

after using

Procedure for Mixing Base Fluids with Sediments

EPA Method 1646	§435.13, §435.15

	Formation oil	Reverse Phase Extraction (RPE) Method for Detection of
Oil Contamination in Non-Aqueous Drilling Fluids (NAF)

EPA Method 1670

Which can be confirmed by :

Determination of Crude Oil Contamination in Non-Aqueous Drilling Fluids
by Gas Chromatography/Mass Spectrometry (GC/MS)

EPA Method 1655	§435.13, §435.15

	Base fluid retained on cuttings	Determination of the Amount of
Non-Aqueous Drilling Fluid (NAF) Base Fluid from Drill Cuttings by a
Retort Chamber (Derived from API Recommended Practice 13B–2)

EPA Method 1674	§435.13, §435.15

Drill cuttings associated with non-aqueous drilling fluids:  Stock
limitations (C16 –C18internal olefin)	Mercury	40 CFR Part 136
§435.13, §435.15

	Cadmium	40 CFR Part 136	§435.13, §435.15

	Polynuclear Aromatic Hydrocarbons (PAH)	PAH Content of Oil by HPLC/UV

EPA Method 1654, Revision A	§435.13, §435.15

	Base fluid sediment toxicity (10-Day Test)	Standard Guide for
Conducting 10-day Static Sediment Toxicity Tests with Marine and
Estuarine Amphipods ASTM E 1367–92

after using

Procedure for Mixing Base Fluids with Sediments

EPA Method 1646	§435.13, §435.15

	Biodegradation rate	Protocol for the Determination of Degradation of
Non-Aqueous Base Fluids in a Marine Closed Bottle Biodegradation Test
System: Modified ISO 11734:1995

EPA Method 1647	§435.13, §435.15

Well treatment, completion, and workover fluids	Oil and grease	40 CFR
Part 136	§435.13, §435.15

	Free oil	Static Sheen Test

EPA Method 1617	§435.12, §435.14

Deck drainage	Free oil	Visual sheen	§435.12, §435.13, §435.14,
§435.15

Domestic waste	Foam	Observation	§435.13, §435.15

	Floating solids	Observation	§435.14, §435.15

	All other domestic waste	See 33 CFR part 151 ‡	§435.14, §435.15

Sanitary M10	Total residual chlorine 	40 CFR Part 136	§435.12,
§435.14, §435.15

Sanitary M9IM	Floating solids	Observation	§435.12, §435.14, §435.15

§435.12 – Off shore BPT

§435.13 – Offshore BAT	§435.14 – Offshore BCT

§435.15 – Offshore NSPS

† There is no discharge of diesel oil (see §435.13 and §435.15).
Diesel oil refers to the grade of distillate fuel oil, as specified in
ASTM D975-91.

‡ This standard references a U.S. Coast Guard regulation: Vessels
Carrying Oil, Noxious Liquid Substances, Garbage, Municipal or
Commercial Waste, And Ballast Water (33 CFR part 151).

Note: The Offshore Subcategory bans the discharge of the following waste
sources and thus there are no related analytical or test methods:
non-aqueous drilling fluids (NAFs) and produced sand (see §435.12,
§435.13, §435.14, §435.15).

. Methods by Waste and Pollutant, Subpart D − Coastal Subcategory

Waste source	Regulated Pollutant Parameter	Analytical/Test Method	Cited
in 40 CFR Part

Produced Water	Oil & grease	40 CFR Part 136	§435.42, §435.43,
§435.44, §435.45

Water-based drilling fluids, drill cuttings, and dewatering effluent	SPP
Toxicity	Drilling Fluids Toxicity Test

EPA Method 1619	§435.43, §435.45

	Free oil	Static Sheen Test

EPA Method 1617	§435.42, §435.43, §435.44, §435.45

	Diesel oil †	ASTM specification D975-91	§435.43, §435.45

	Mercury	40 CFR Part 136	§435.43, §435.45

	Cadmium	40 CFR Part 136	§435.43, §435.45

Well treatment, workover, and completion fluids	Oil and grease	40 CFR
Part 136	§435.43, §435.45

	Free oil	Static Sheen Test

EPA Method 1617	§435.42, §435.44

Deck drainage	Free oil	Visual sheen	§435.42,§435.43, §435.44,
§435.45

Domestic waste	Foam	Observation	§435.43, §435.45

	Floating solids and garbage	Observation	§435.42, §435.44, §435.45

Sanitary M10	Total residual chlorine 	40 CFR Part 136	§435.42,
§435.44, §435.45

Sanitary M9IM	Floating solids	Observation	§435.42, §435.44, §435.45

§435.42 – Coastal BPT

§435.43 – Coastal BAT	§435.44 – Coastal BCT

§435.45 – Coastal NSPS

† There is no discharge of diesel oil (see §435.43 and §435.45).
Diesel oil refers to the grade of distillate fuel oil, as specified in
ASTM D975-91.

Notes:

(1) The Coastal Subcategory bans the discharge of the following waste
sources and thus there are no related analytical or test methods:
non-aqueous drilling fluids (NAFs) and produced sand (see §435.42,
§435.43, §435.44, §435.45).

(2) The Coastal Subcategory bans the discharge of drill cuttings
associated with non-aqueous drilling fluids unless there are technical
limitations (see §435.43 and §435.45). Appendix 1 of 40 CFR Part 435
Subpart D provides the method for permit writers to determine when
operators qualify for an exemption from this discharge prohibition. For
those operators that quality for the exemption from the discharge
prohibition EPA requires that these operators meet the same stock
limitations and discharge limitations for drill cuttings associated with
non-aqueous drilling fluids for operators in Offshore waters (see 40 CFR
§435.13 and §435.15) and no discharge of free oil (determined by the
Static Sheen Test, required by §435.42, §435.44) in order to discharge
drill cuttings associated with non-aqueous drilling fluids.

Static Sheen Test (EPA Method 1617)

Scope and Application

This method is to be used as a compliance test for the “no discharge
of free oil” requirement for discharges of drilling fluids, drill
cuttings, and well treatment, completion and workover fluids. “Free
oil” refers to any oil contained in a waste stream that when
discharged will cause a film or sheen upon or a discoloration of the
surface of the receiving water.

Summary of Method

Samples (15-mL) of drilling fluids or well treatment, completion, and
workover fluids, and 15-g samples (wet weight basis) of drill cuttings
or produced sand are introduced into ambient seawater in a container
having an air-to-liquid interface area of 1,000 cm2 (155.5 in2). Samples
are dispersed within the container and observations made no more than
one hour later to ascertain if these materials cause sheen, iridescence,
gloss, or increased reflectance on the surface of the test seawater. The
occurrence of any of these visual observations will constitute a
demonstration that the tested material contains “free oil,” and
therefore results in a prohibition of its discharge into receiving
waters.

Drilling Fluids Toxicity Test (EPA Method 1619)

Scope and Application

This method is to be used as a compliance test for the suspended
particulate phase (SPP) toxicity requirement for discharges of
water-based drilling fluids, associated drill cuttings, and dewatering
effluent and for drill cuttings associated with non–aqueous drilling
fluids. The test may be conducted as a full bioassay to estimate the
concentration that is lethal to 50% of the test organisms (LC50) or as a
partial test to determine compliance with 40 CFR Part 435 requirements.

Summary of Method

Samples of drilling fluids or drilling mud from active field systems are
diluted with natural or artificial seawater, mixed, and settled for 1
hour. The suspended particulate phase (SPP) is decanted. The decanted
solution, defined to be 100% SPP, is further diluted with seawater to
obtain test concentrations. Mysidopsis bahia (mysid shrimp) are exposed
to the test concentrations for 96 hours, at which time the living
organisms are counted. For the full bioassay, a range of concentrations
is tested and the LC50 is calculated. For the partial test, a negative
control, 3% concentration, and positive control are tested. If the
number of organisms killed in the 3% test concentration meets a number
specified in the method, the test material passes the partial toxicity
test.

Procedure for Mixing Base Fluids with Sediments (EPA Method 1646)

This method is used to amend 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 are press-sieved through a 2,000 micron
mesh sieve to remove large debris. Then the control sediments are
press-sieved through a 500 micron sieve to remove indigenous organisms
that may prey on the test species or otherwise confound test results.
The control sediment is homogenized to limit the effects of settling
that may have occurred during storage. The control sediments are
homogenized before density determinations and before the addition of
base fluid. Because base fluids are 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).

Protocol for the Determination of Degradation of Non Aqueous Base Fluids
in a Marine Closed Bottle Biodegradation Test System: Modified ISO
11734:1995 (EPA Method 1647)

Scope and Application

EPA promulgated the use of the marine anaerobic biodegradation analytic
method because it most closely modeled the ability of a drilling fluid
to biodegrade anaerobically in marine environments (January 22, 2001; 66
FR 6864). This method determines the anaerobic degradation potential of
mineral oils, paraffin oils and non aqueous fluids (NAF) in sediments.
These substrates are base fluids for formulating offshore drilling
fluids. The test evaluates base fluid biodegradation rates by monitoring
headspace gas production due to microbial degradation of the test fluid
in natural marine sediment.

Subsequent to this promulgation, EPA incorporated additional quality
assurance procedures for the marine anaerobic biodegradation analytic
method in the NPDES permit for the Western Gulf of Mexico (“Final
NPDES General Permit for New and Existing Sources and New Dischargers in
the Offshore Subcategory of the Oil and Gas Extraction Category for the
Western Portion of the Outer Continental Shelf of the Gulf of Mexico,”
GMG290000, Appendix B).  The additional quality assurance instructions
in the GMG290000 more clearly describe the sample preparation and
compliance determination steps.  Specifically, these additional quality
assurance procedures clarify that users must only use headspace gas to
determine compliance with the Part 435 effluent guidelines. EPA
solicited public comment on these additional quality assurance
procedures in the proposed 2010 Method Update Rule (XXX – insert date
and FR citation for proposed rule) before publication of the final 2010
Method Update Rule (XXX – insert date and FR citation for final rule).

Summary of Method

A mixture of marine/estuarine sediment, test substrate (hydrocarbon or
controls), and seawater is placed into clean 120 ml (150 ml actual
volume) Wheaton serum bottles. The test is run using four replicate
serum bottles containing 2,000 mg carbon/kg dry weight concentration of
test substrate in sediment. The anaerobic (redox) condition of the
bottles is evaluated using resazurin dye solution (dye is blue when
oxygen is present, reddish in low oxygen conditions and colorless if
oxygen free). Headspace air is removed with a nitrogen sparge before
incubation begins. Gas production and composition are measured
approximately every two weeks. Gas production is measured using a
pressure gauge. Barometric pressure is measured at the time of testing
to make necessary volume adjustments. The test period is 275 days. The
results of EPA Method 1647 for the test fluid are compared against the
reference fluid to determine whether the test fluid is eligible for
discharge with drill cuttings.

Determination of Crude Oil Contamination in Non-Aqueous Drilling Fluids
by Gas Chromatography/Mass Spectrometry (GC/MS) (EPA Method 1655)

Scope and Application

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

Summary of Method

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

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. 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. 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.

Reverse Phase Extraction (RPE) Method for Detection of Oil Contamination
in Non-Aqueous Drilling Fluids (NAF) (EPA Method 1670)

Scope and Application

This method is used for determination of crude or formation oil, or
other petroleum oil contamination, in non-aqueous drilling fluids
(NAFs). It is intended as a positive/negative test to determine a
presence of crude oil in NAF prior to discharging drill cuttings from
offshore production platforms. It is for use in the Environmental
Protection Agency's (EPA's) survey and monitoring programs under the
Clean Water Act, including monitoring of compliance with the Gulf of
Mexico NPDES General Permit for monitoring of oil contamination in
drilling fluids. The method has been designed to show positive
contamination for 5% of representative crude oils at a concentration of
0.1% in drilling fluid (vol/vol), 50% of representative crude oils at a
concentration of 0.5%, and 95% of representative crude oils at a
concentration of 1%.

Summary of Method

An aliquot of drilling fluid is extracted using isopropyl alcohol. The
mixture is allowed to settle and then filtered to separate out residual
solids. An aliquot of the filtered extract is charged onto a reverse
phase extraction (RPE) cartridge. The cartridge is eluted with isopropyl
alcohol. Crude oil contaminates are retained on the cartridge and their
presence (or absence) is detected based on observed fluorescence using a
black light.

Determination of the Amount Of Non-Aqueous Drilling Fluid (NAF) Base
Fluid from Drill Cuttings by a Retort Chamber (Derived From API
Recommended Practice 13B–2) (EPA Method 1674)

Scope and Application

This procedure is specifically intended to measure the amount of
non-aqueous drilling fluid (NAF) base fluid from cuttings generated
during a drilling operation. This procedure is a retort test which
measures all oily material (NAF base fluid) and water released from a
cuttings sample when heated in a calibrated and properly operating
“Retort” instrument.

Summary of Method

A known mass of cuttings is heated in the retort chamber to vaporize the
liquids associated with the sample. The NAF base fluid and water vapors
are then condensed, collected, and measured in a precision graduated
receiver.

PAH Content of Oil by HPLC/UV (EPA Method 1654, Revision A)

Scope and Application

This method is designed to determine the polynuclear aromatic
hydrocarbon (PAH) content of oil by high-performance liquid
chromatography (HPLC) with an ultra-violet absorption (UV) detector. The
PAH content is measured and reported as phenanthrene. For oil in
drilling muds, this method is designed to be used in conjunction with
the extraction procedure in EPA Method 1662.

Summary of Method

cetonitrile and a 20-μL aliquot is injected into the HPLC. The

PAHs are partially separated by HPLC and detected with the UV detector.
Identification of PAH (qualitative analysis) is performed by comparing
the response of the UV detector to the response during the
retention-time range characteristic of the PAH in diesel oil. PAH is
present when a response occurs during this retention-time range.
Quantitative analysis is performed by calibrating the HPLC with
phenanthrene using an external standard technique, and using the
calibration factor to determine the concentration of PAH in the sample.

Previous Publication of Oil and Gas Extraction Point Source Category
Analytic Methods

EPA previously published the analytical methods for the Oil and Gas
Extraction Point Source Category in the Code of Federal Regulations
(CFR). These methods were published as attachments to Subpart A
(Offshore Subcategory). Below is the listing of these methods as they
appeared in the CFR and their current EPA method number. The 2010 Method
Update rule deleted the full publication of the Oil and Gas Extraction
analytical methods from the CFR and incorporated these methods by
reference (XXX – insert date and FR citation for final rule).

Table   STYLEREF 1 \s  1 -  SEQ Table \* ARABIC \s 1  3 . EPA Method
Numbers for Oil and Gas Extraction Point Source Category Analytical
Methods and Prior CFR References

Analytical/Test Method	EPA Method Number	Date First Promulgated	Previous
CFR References

Static Sheen Test	1617	1993	Subpart A, Appendix 1

Drilling Fluids Toxicity Test 	1619	1993	Subpart A, Appendix 2

Procedure for Mixing Base Fluids With Sediments	1646	2001	Subpart A,
Appendix 3

Protocol for the Determination of Degradation of Non Aqueous Base Fluids
in a Marine Closed Bottle Biodegradation Test System: Modified ISO
11734:1995 †	1647	2001	Subpart A, Appendix 4

Determination of Crude Oil Contamination in Non-Aqueous Drilling Fluids
by Gas Chromatography/Mass Spectrometry (GC/MS)	1655	2001	Subpart A,
Appendix 5

Reverse Phase Extraction (RPE) Method for Detection of Oil Contamination
in Non-Aqueous Drilling Fluids (NAF)	1670	2001	Subpart A, Appendix 6

Determination of the Amount of Non-Aqueous Drilling Fluid (NAF) Base
Fluid from Drill Cuttings by a Retort Chamber (Derived from API
Recommended Practice 13B–2)	1674	2001	Subpart A, Appendix 7

† EPA included additional quality control measures into the marine
closed bottle biodegradation test in this document from EPA’s Western
Gulf of Mexico Offshore General NPDES Permit, No. GMG 290000. EPA
solicited public comment on these additional quality assurance
procedures in the proposed 2010 Method Update Rule (XXX – insert date
and FR citation for proposed rule) before publication of the final 2010
Method Update Rule (XXX – insert date and FR citation for final rule).

Static Sheen Test (EPA Method 1617)

Scope and Application

This method is to be used as a compliance test for the “no discharge
of free oil” requirement for discharges of drilling fluids, drill
cuttings, produced sand, and well treatment, completion and workover
fluids. “Free oil” refers to any oil contained in a waste stream
that when discharged will cause a film or sheen upon or a discoloration
of the surface of the receiving water.

Summary of Method

15-mL samples of drilling fluids or well treatment, completion, and
workover fluids, and 15-g samples (wet weight basis) of drill cuttings
or produced sand are introduced into ambient seawater in a container
having an air-to-liquid interface area of 1,000 cm2 (155.5 in2). Samples
are dispersed within the container and observations made no more than
one hour later to ascertain if these materials cause a sheen,
iridescence, gloss, or increased reflectance on the surface of the test
seawater. The occurrence of any of these visual observations will
constitute a demonstration that the tested material contains “free
oil,” and therefore results in a prohibition of its discharge into
receiving waters.

Interferences

Residual “free oil” adhering to sampling containers, the magnetic
stirring bar used to mix the sample, and the stainless steel spatula
used to mix the sample will be the principal sources of contamination
problems. These problems should only occur if improperly washed and
cleaned equipment are used for the test. The use of disposable equipment
minimizes the potential for similar contamination from pipettes and the
test container.

Apparatus, Materials, and Reagents

Apparatus

Sampling Containers: 1-liter polyethylene beakers and 1-liter glass
beakers.

Graduated cylinder: 100-mL graduated cylinder required only for
operations where predilution of mud discharges is required.

Plastic disposable weighing boats.

Triple-beam scale.

Disposable pipettes: 25-mL disposable pipettes.

Magnetic stirrer and stirring bar.

Stainless steel spatula.

Test container: Open plastic container whose internal cross-section
parallel to its opening has an area of 1,000 cm2  ±50 cm2 (155.5 ±7.75
in2), and a depth of at least 13 cm (5 inches) and no more than 30 cm
(11.8 inches).

Materials and Reagents.

Plastic liners for the test container: Oil-free, heavy-duty plastic
trash can liners that do not inhibit the spreading of an oil film.
Liners must be of sufficient size to completely cover the interior
surface of the test container. Permittees must determine an appropriate
local source of liners that do not inhibit the spreading of 0.05 mL of
diesel fuel added to the lined test container under the test conditions
and protocol described below.

Ambient receiving water.

Calibration

None currently specified.

Quality Control Procedures

None currently specified.

Sample Collection and Handling

Sampling containers must be thoroughly washed with detergent, rinsed a
minimum of three times with fresh water, and allowed to air dry before
samples are collected.

Samples of drilling fluid to be tested shall be taken at the shale
shaker after cuttings have been removed. The sample volume should range
between 200 mL and 500 mL.

Samples of drill cuttings will be taken from the shale shaker screens
with a clean spatula or similar instrument and placed in a glass beaker.
Cuttings samples shall be collected prior to the addition of any
washdown water and should range between 200 g and 500 g.

Samples of produced sand must be obtained from the solids control
equipment from which the discharge occurs on any given day and shall be
collected prior to the addition of any washdown water; samples should
range between 200 g and 500 g.

Samples of well treatment, completion, and workover fluids must be
obtained from the holding facility prior to discharge; the sample volume
should range between 200 mL and 500 mL.

Samples must be tested no later than 1 hour after collection.

Drilling fluid samples must be mixed in their sampling containers for 5
minutes prior to the test using a magnetic bar stirrer. If predilution
is imposed as a permit condition, the sample must be mixed at the same
ratio with the same prediluting water as the discharged muds and stirred
for 5 minutes.

Drill cuttings must be stirred and well mixed by hand in their sampling
containers prior to testing, using a stainless steel spatula.

Procedure

Ambient receiving water must be used as the “receiving water” in the
test. The temperature of the test water shall be as close as practicable
to the ambient conditions in the receiving water, not the room
temperature of the observation facility. The test container must have an
air-to-liquid interface area of 1,000  ±50 cm2. The surface of the
water should be no more than 1.27 cm (0.5 inch) below the top of the
test container.

Plastic liners shall be used, one per test container, and discarded
afterwards. Some liners may inhibit spreading of added oil; operators
shall determine an appropriate local source of liners that do not
inhibit the spreading of the oil film.

A 15-mL sample of drilling fluid or well treatment, completion, and
workover fluids must be introduced by pipette into the test container 1
cm below the water surface. Pipettes must be filled and discharged with
test material prior to the transfer of test material and its
introduction into test containers. The test water/test material mixture
must be stirred using the pipette to distribute the test material
homogeneously throughout the test water. The pipette must be used only
once for a test and then discarded.

Drill cuttings or produced sand should be weighed on plastic weighing
boats; 15-g samples must be transferred by scraping test material into
the test water with a stainless steel spatula. Drill cuttings shall not
be prediluted prior to testing. Also, drilling fluids and cuttings will
be tested separately. The weighing boat must be immersed in the test
water and scraped with the spatula to transfer any residual material to
the test container. The drill cuttings or produced sand must be stirred
with the spatula to an even distribution of solids on the bottom of the
test container.

Observations must be made no later than 1 hour after the test material
is transferred to the test container. Viewing points above the test
container should be made from at least three sides of the test
container, at viewing angles of approximately 60° and 30° from the
horizontal. Illumination of the test container must be representative of
adequate lighting for a working environment to conduct routine
laboratory procedures. It is recommended that the water surface of the
test container be observed under a fluorescent light source such as a
dissecting microscope light. The light source shall be positioned above
and directed over the entire surface of the pan.

Detection of a “silvery” or “metallic” sheen or gloss, increased
reflectivity, visual color, iridescence, or an oil slick on the water
surface of the test container surface shall constitute a demonstration
of “free oil.” These visual observations include patches, streaks,
or sheets of such altered surface characteristics. If the free oil
content of the sample approaches or exceeds 10%, the water surface of
the test container may lack color, a sheen, or iridescence, due to the
increased thickness of the film; thus, the observation for an oil slick
is required. The surface of the test container shall not be disturbed in
any manner that reduces the size of any sheen or slick that may be
present.

If an oil sheen or slick occurs on less than one-half of the surface
area after the sample is introduced to the test container, observations
will continue for up to 1 hour. If the sheen or slick increases in size
and covers greater than one-half of the surface area of the test
container during the observation period, the discharge of the material
shall cease. If the sheen or slick does not increase in size to cover
greater than one-half of the test container surface area after one hour
of observation, discharge may continue and additional sampling is not
required.

If a sheen or slick occurs on greater than one-half of the surface area
of the test container after the test material is introduced, discharge
of the tested material shall cease. The permittee may retest the
material causing the sheen or slick. If subsequent tests do not result
in a sheen or slick covering greater than one-half of the surface area
of the test container, discharge may continue.

Drilling Fluids Toxicity Test (EPA Method 1619)

Scope and Application

This method is to be used as a compliance test for the suspended
particulate phase (SPP) toxicity requirement for discharges of
water-based drilling fluids, associated drill cuttings, and dewatering
effluent and for drill cuttings associated with non –aqueous drilling
fluids. The test may be conducted as a full bioassay to estimate the
concentration that is lethal to 50% of the test organisms (LC50) or as a
partial test to determine compliance with 40 CFR Part 435 requirements.

Summary of Method

Samples of drilling fluids or drilling mud from active field systems are
diluted with natural or artificial seawater, mixed, and settled for 1
hour. The suspended particulate phase (SPP) is decanted. The decanted
solution, defined to be 100% SPP, is further diluted with seawater to
obtain test concentrations. Mysidopsis bahia (mysid shrimp) are exposed
to the test concentrations for 96 hours, at which time the living
organisms are counted. For the full bioassay, a range of concentrations
is tested and the LC50 is calculated. For the partial test, a negative
control, 3% concentration, and positive control are tested. If the
number of organisms killed in the 3% test concentration meets a number
specified in the method, the test material passes the partial toxicity
test.

Sample Collection

The collection and preservation methods for drilling fluids (muds) and
water samples presented here are designed to minimize sample
contamination and alteration of the physical or chemical properties of
the samples due to freezing, air oxidation, or drying.

Apparatus

The following items are required for water and drilling mud sampling and
storage:

Acid-rinsed linear-polyethylene bottles or other appropriate
noncontaminating drilling mud sampler.

Acid-rinsed linear-polyethylene bottles or other appropriate
noncontaminating water sampler.

Acid-rinsed linear-polyethylene bottles or other appropriate
noncontaminated vessels for water and mud samples.

Ice chests for preservation and shipping of mud and water samples.

Water Sampling

Collection of water samples shall be made with appropriate acid-rinsed
linear-polyethylene bottles or other appropriate non-contaminating water
sampling devices. Special care shall be taken to avoid the introduction
of contaminants from the sampling devices and containers. Prior to use,
the sampling devices and containers should be thoroughly cleaned with a
detergent solution, rinsed with tap water, soaked in 10% hydrochloric
acid (HCl) for 4 hours, and then thoroughly rinsed with glass-distilled
water.

Drilling Mud Sampling

Drilling mud formulations to be tested shall be collected from active
field systems. Obtain a well-mixed sample from beneath the shale shaker
after the mud has passed through the screens. Samples shall be stored in
polyethylene containers or in other appropriate uncontaminated vessels.
Prior to sealing the sample containers on the platform, flush as much
air out of the container by filling it with drilling fluid sample,
leaving a one inch space at the top.

Mud samples shall be immediately shipped to the testing facility on blue
or wet ice (do not use dry ice) and continuously maintained at 0–4°C
until the time of testing.

Bulk mud samples shall be thoroughly mixed in the laboratory using a
1,000 rpm high shear mixer and then subdivided into individual, small
wide-mouthed (e.g., one or two liter) non-contaminating containers for
storage.

The drilling muds stored in the laboratory shall have any excess air
removed by flushing the storage containers with nitrogen under pressure
anytime the containers are opened. Moreover, the sample in any container
opened for testing must be thoroughly stirred using a 1,000 rpm high
shear mixer prior to use.

Most drilling mud samples may be stored for periods of time longer than
2 weeks prior to toxicity testing provided that proper containers are
used and proper condition are maintained.

Suspended Particulate Phase Sample Preparation

Mud samples that have been stored under specified conditions in this
protocol shall be prepared for tests within three months after
collection. The SPP shall be prepared as detailed below.

Apparatus

The following items are required:

Magnetic stir plates and bars.

Several graduated cylinders, ranging in volume from 10 mL to 1 L

Large (15 cm) powder funnels.

Several 2-liter graduated cylinders.

Several 2-liter large mouth graduated Erlenmeyer flasks.

Prior to use, all glassware shall be thoroughly cleaned. Wash all
glassware with detergent, rinse five times with tap water, rinse once
with acetone, rinse several times with distilled or deionized water,
place in a clean 10% (or stronger) HCl acid bath for a minimum of 4
hours, rinse five times with tap water, and then rinse five times with
distilled or deionized water. For test samples containing mineral oil or
diesel oil, glassware should be washed with petroleum ether to assure
removal of all residual oil.

Note: If the glassware with nytex cups soaks in the acid solution longer
than 24 hours, then an equally long deionized water soak should be
performed.

Test Seawater Sample Preparation

Diluent seawater and exposure seawater samples are prepared by
filtration through a 1.0 micrometer filter prior to analysis.

Artificial seawater may be used as long as the seawater has been
prepared by standard methods or ASTM methods, has been properly
“seasoned,” filtered, and has been diluted with distilled water to
the same specified 20 ±2 ppt salinity and 20 ±2°C temperature as
the “natural” seawater.

Sample Preparation

The pH of the mud shall be tested prior to its use. If the pH is less
than 9, if black spots have appeared on the walls of the sample
container, or if the mud sample has a foul odor, that sample shall be
discarded. Subsample a manageable aliquot of mud from the well-mixed
original sample. Mix the mud and filtered test seawater in a volumetric
mud-to-water ratio of 1 to 9. This is best done by the method of
volumetric displacement in a 2–L, large mouth, graduated Erlenmeyer
flask. Place 1,000 mL of seawater into the graduated Erlenmeyer flask.
The mud subsample is then carefully added via a powder funnel to obtain
a total volume of 1200 mL. (A 200 mL volume of the mud will now be in
the flask).

The 2–L, large mouth, graduated Erlenmeyer flask is then filled to the
2,000 mL mark with 800 mL of seawater, which produces a slurry with a
final ratio of one volume drilling mud to nine volumes water. If the
volume of SPP required for testing or analysis exceeds 1,500 to 1,600
mL, the initial volumes should be proportionately increased.
Alternatively, several 2–L drill mud/water slurries may be prepared as
outlined above and combined to provide sufficient SPP.

Mix this mud/water slurry with magnetic stirrers for 5 minutes. Measure
the pH and, if necessary, adjust (decrease) the pH of the slurry to
within 0.2 units of the seawater by adding 6N HCl while stirring the
slurry. Then, allow the slurry to settle for 1 hour. Record the amount
of HCl added.

At the end of the settling period, carefully decant (do not siphon) the
Suspended Particulate Phase (SPP) into an appropriate container.
Decanting the SPP is one continuous action. In some cases no clear
interface will be present; that is, there will be no solid phase that
has settled to the bottom. For those samples the entire SPP solution
should be used when preparing test concentrations. However, in those
cases when no clear interface is present, the sample must be remixed for
five minutes. This insures the homogeneity of the mixture prior to the
preparation of the test concentrations. In other cases, there will be
samples with two or more phases, including a solid phase. For those
samples, carefully and continuously decant the supernatant until the
solid phase on the bottom of the flask is reached. The decanted solution
is defined to be 100% SPP. Any other concentration of SPP refers to a
percentage of SPP that is obtained by volumetrically mixing 100% SPP
with seawater.

SPP samples to be used in toxicity tests shall be mixed for 5 minutes
and must not be preserved or stored.

Measure the filterable and unfilterable residue of each SPP prepared for
testing. Measure the dissolved oxygen (DO) and pH of the SPP. If the DO
is less than 4.9 ppm, aerate the SPP to at least 4.9 ppm which is 65% of
saturation. Maximum allowable aeration time is 5 minutes using a generic
commercial air pump and air stone. Neutralize the pH of the SPP to a pH
7.8 ±0.1 using a dilute HCl solution. If too much acid is added to
lower the pH saturated NaOH may be used to raise the pH to 7.8 ±0.1
units. Record the amount of acid or NaOH needed to lower/raise to the
appropriate pH. Three repeated DO and pH measurements are needed to
insure homogeneity and stability of the SPP. Preparation of test
concentrations may begin after this step is complete.

Add the appropriate volume of 100% SPP to the appropriate volume of
seawater to obtain the desired SPP concentration. The control is
seawater only. Mix all concentrations and the control for 5 minutes by
using magnetic stirrers. Record the time; and, measure DO and pH for Day
0. Then, the animals shall be randomly selected and placed in the dishes
in order to begin the 96-hour toxicity test.

Guidance for Performing Suspended Particulate Phase Toxicity Tests Using
Mysidopsis bahia

Apparatus

Items listed by Borthwick [1] are required for each test series, which
consists of one set of control and test containers, with three
replicates of each.

Sample Collection Preservation

Drilling muds and water samples are collected and stored as described in
Section   REF _Ref239147265 \r \h  \* MERGEFORMAT  3.3.3 .

Species Selection

The Suspended Particulate Phase (SPP) tests on drilling muds shall
utilize the test species Mysidopsis bahia. Test animals shall be 3 to 6
days old on the first day of exposure. Whatever the source of the
animals, collection and handling should be as gentle as possible.
Transportation to the laboratory should be in well-aerated water from
the animal culture site at the temperature and salinity from which they
were cultured. Methods for handling, acclimating, and sizing bioassay
organisms given by Borthwick [1] and Nimmo [2] shall be followed in
matters for which no guidance is given here.

Experimental Conditions

Suspended particulate phase (SPP) tests should be conducted at a
salinity of 20 ±2 ppt. Experimental temperature should be 20 ±2°C.
Dissolved oxygen in the SPP shall be raised to or maintained above 65%
of saturation prior to preparation of the test concentrations. Under
these conditions of temperature and salinity, 65% saturation is a DO of
5.3 ppm. Beginning at Day 0-before the animals are placed in the test
containers DO, temperature, salinity, and pH shall be measured every 24
hours. DO should be reported in milligrams per liter.

Aeration of test media is required during the entire test with a rate
estimated to be 50–140 cubic centimeters/minute. This air flow to each
test dish may be achieved through polyethylene tubing (0.045-inch inner
diameter and 0.062-inch outer diameter) by a small generic aquarium
pump. The delivery method, surface area of the aeration stone, and flow
characteristics shall be documented. All treatments, including control,
shall be the same.

Light intensity shall be 1,200 microwatts/cm2 using cool white
fluorescent bulbs with a 14-hr light and 10-hr dark cycle. This
light/dark cycle shall also be maintained during the acclimation period
and the test.

Experimental Procedure

Wash all glassware with detergent, rinse five times with tap water,
rinse once with acetone, rinse several times with distilled or deionized
water, place in a clean 10% HCl acid bath for a minimum of 4 hours,
rinse five times with tap water, and then rinse five times with
distilled water.

Establish the definitive test concentration based on results of a range
finding test. A minimum of five test concentrations plus a negative and
positive (reference toxicant) control is required for the definitive
test. To estimate the LC50, two concentrations shall be chosen that give
(other than zero and 100%) mortality above and below 50%.

Twenty organisms are exposed in each test dish. Nytex® cups shall be
inserted into every test dish prior to adding the animals. These
“nylon mesh screen” nytex holding cups are fabricated by gluing a
collar of 363-micrometer mesh nylon screen to a 15-centimeter wide Petri
dish with silicone sealant. The nylon screen collar is approximately 5
centimeters high. The animals are then placed into the test
concentration within the confines of the Nytex cups.

Individual organisms shall be randomly assigned to treatment. A
randomization procedure is presented in Section   REF _Ref239147405 \r
\h  \* MERGEFORMAT  3.7 . Make every attempt to expose animals of
approximately equal size. The technique described by Borthwick [1], or
other suitable substitutes, should be used for transferring specimens.
Throughout the test period, mysids shall be fed daily with approximately
50 Artemia (brine shrimp) nauplii per mysid. This will reduce stress and
decrease cannibalism.

Cover the dishes, aerate, and incubate the test containers in an
appropriate test chamber. Positioning of the test containers holding
various concentrations of test solution should be randomized if
incubator arrangement indicates potential position difference. The test
medium is not replaced during the 96-hour test.

Observations may be attempted at 4, 6 and 8 hours; they must be
attempted at 0, 24, 48, and 72 hours and must be made at 96 hours.
Attempts at observations refers to placing a test dish on a light table
and visually counting the animals. Do not lift the “nylon mesh
screen” cup out of the test dish to make the observation. No
unnecessary handling of the animals should occur during the 96 hour test
period. DO and pH measurements must also be made at 0, 24, 48, 72, and
96 hours. Take and replace the test medium necessary for the DO and pH
measurements outside of the nytex cups to minimize stresses on the
animals.

At the end of 96 hours, all live animals must be counted. Death is the
end point, so the number of living organisms is recorded. Death is
determined by lack of spontaneous movement. All crustaceans molt at
regular intervals, shedding a complete exoskeleton. Care should be taken
not to count an exoskeleton. Dead animals might decompose or be eaten
between observations. Therefore, always count living, not dead animals.
If daily observations are made, remove dead organisms and molted
exoskeletons with a pipette or forceps. Care must be taken not to
disturb living organisms and to minimize the amount of liquid withdrawn.

Methods for Positive Control Tests (Reference Toxicant)

Sodium lauryl sulfate (dodecyl sodium sulfate) is used as a reference
toxicant for the positive control. The chemical used should be
approximately 95% pure. The source, lot number, and percent purity shall
be reported.

Test methods are those used for the drilling fluid tests, except that
the test material was prepared by weighing one gram sodium lauryl
sulfate on an analytical balance, adding the chemical to a
100-milliliter volumetric flask, and bringing the flask to volume with
deionized water. After mixing this stock solution, the test mixtures are
prepared by adding 0.1 milliliter of the stock solution for each part
per million desired to one liter of seawater.

The mixtures are stirred briefly, water quality is measured, animals are
added to holding cups, and the test begins. Incubation and monitoring
procedures are the same as those for the drilling fluids.

Randomization Procedure

Purpose and Procedure

The purpose of this procedure is to assure that mysids are impartially
selected and randomly assigned to six test treatments (five drilling
fluid or reference toxicant concentrations and a control) and
impartially counted at the end of the 96-hour test. Thus, each test
setup, as specified in the randomization procedure, consists of 3
replicates of 20 animals for each of the six treatments, i.e., 360
animals per test.   REF _Ref239147452 \h  Figure 3-1  is a flow diagram
that depicts the procedure schematically and should be reviewed to
understand the over-all operation. The following tasks shall be
performed in the order listed.

Mysids are cultured in the laboratory in appropriate units. If mysids
are purchased, go to Task 3 (Section   REF _Ref239147716 \r \h  <IP_ADDRESS>
).

Remove mysids from culture tanks (6, 5, 4, and 3 days before the test
will begin, i.e., Tuesday, Wednesday, Thursday, and Friday if the test
will begin on Monday) and place them in suitably large maintenance
containers so that they can swim about freely and be fed.

Note: Not every detail (the definition of suitably large containers, for
example) is provided here. Training and experience in aquatic animal
culture and testing will be required to successfully complete these
tests.

Figure   STYLEREF 1 \s  3 -  SEQ Figure \* ARABIC \s 1  1 . Mysid
Randomization Procedure

Remove mysids from maintenance containers and place all animals in a
single container. The intent is to have homogeneous test population of
mysids of a known age (3–6 days old).

For each toxicity test, assign two suitable containers (500-milliliter
(mL) beakers are recommended) for mysid separation/enumeration. Label
each container (A1, A2, B1, B2, and C1, C2, for example, if two drilling
fluid tests and a reference toxicant test are to be set up on one day).
The purpose of this task is to allow the investigator to obtain a close
estimate of the number of animals available for testing and to prevent
unnecessary crowding of the mysids while they are being counted and
assigned to test containers. Transfer the mysids from the large test
population container to the labeled separation and enumeration
containers but do not place more than 200 mysids in a 500-mL beaker. Be
impartial in transferring the mysids; place approximately equal numbers
of animals (10–15 mysids is convenient) in each container in a cyclic
manner rather than placing the maximum number each container at one
time.

Note: It is important that the animals not be unduly stressed during
this selection and assignment procedure. Therefore, it will probably be
necessary to place all animals (except the batch immediately being
assigned to test containers) in mesh cups with flowing seawater or in
large volume containers with aeration. The idea is to provide the
animals with near optimal conditions to avoid additional stress.

Place the mysids from the two labeled enumeration containers assigned to
a specific test into one or more suitable containers to be used as
counting dishes (2-liter Carolina dishes are suggested). Because of the
time required to separate, count, and assign mysids, two or more people
may be involved in completing this task. If this is done, two or more
counting dishes may be used, but the investigator must make sure that
approximately equal numbers of mysids from each labeled container are
placed in each counting dish.

By using a large-bore, smooth-tip glass pipette, select mysids from the
counting dish(es) and place them in the 36 individually numbered
distribution containers (10-ml beakers are suggested). The mysids are
assigned two at a time to the 36 containers by using a randomization
schedule similar to the one presented below. At the end of
selection/assignment round 1, each container will contain two mysids; at
the end of round 2, they will contain four mysids; and so on until each
contains ten mysids.

Table   STYLEREF 1 \s  3 -  SEQ Table \* ARABIC \s 1  1 . Example of a
Randomization Schedule

Selection/assignment round (2 mysids each)	Place mysid in the numbered
distribution containers in the random order shown

1	8, 21, 6, 28, 33, 32, 1, 3, 10, 9, 4, 14, 23, 2, 34, 22, 36, 27, 5,
30, 35, 24, 12, 25, 11, 17, 19, 26, 31, 7, 20, 15, 18, 13, 16, 29.

2	35, 18, 5, 12, 32, 34, 22, 3, 9, 16, 26, 13, 20, 28, 6, 21, 24, 30, 8,
31, 7, 23, 2, 15, 25, 17, 1, 11, 27, 4, 19, 36, 10, 33, 14, 29.

3	7, 19, 14, 11, 34, 21, 25, 27, 17, 18, 6, 16, 29, 2, 32, 10, 4, 20, 3,
9, 1, 5, 28, 24, 31, 15, 22, 13, 33, 26, 36, 12, 8, 30, 35, 23.

4	30, 2, 18, 5, 8, 27, 10, 25, 4, 20, 26, 15, 31, 36, 35, 23, 11, 29,
16, 17, 28, 1, 33, 14, 9, 34, 7, 3, 12, 22, 21, 6, 19, 24, 32, 13.

5	34, 28, 16, 17, 10, 12, 1, 36, 20, 18, 15, 22, 2, 4, 19, 23, 27, 29,
25, 21, 30, 3, 9, 33, 32, 6, 14, 11, 35, 24, 26, 7, 31, 5, 13, 8.

Transfer mysids from the 36 distribution containers to 18 labeled test
containers in random order. A label is assigned to each of the three
replicates (A, B, C) of the six test concentrations. Count and record
the 96 hour response in an impartial order.

Repeat Tasks 5–7 (Sections   REF _Ref239147785 \r \h  \* MERGEFORMAT
<IP_ADDRESS>  through   REF _Ref239147771 \r \h  \* MERGEFORMAT  <IP_ADDRESS> )
for each toxicity test. A new random schedule should be followed in
Tasks 6 and 7 for each test.

Note: If a partial toxicity test is conducted, the procedures described
above are appropriate and should be used to prepare the single test
concentration and control, along with the reference toxicant test.

Data Analysis and Interpretation

Complete survival data in all test containers at each observation time
shall be presented in tabular form. If greater than 10% mortality occurs
in the controls, all data shall be discarded and the experiment
repeated. Unacceptably high control mortality indicates the presence of
important stresses on the organisms other than the material being
tested, such as injury or disease, stressful physical or chemical
conditions in the containers, or improper handling, acclimation, or
feeding. If 10% mortality or less occurs in the controls, the data may
be evaluated and reported.

A definitive, full bioassay conducted according to the EPA protocol is
used to estimate the concentration that is lethal to 50% of the test
organisms that do not die naturally. This toxicity measure is known as
the median lethal concentration, or LC50. The LC50 is adjusted for
natural mortality or natural responsiveness. The maximum likelihood
estimation procedure with the adjustments for natural responsiveness as
given by D.J. Finney, in Probit Analysis 3rd edition, 1971, Cambridge
University Press, chapter 7, can be used to obtain the probit model
estimate of the LC50 and the 95% fiducial (confidence) limits for the
LC50. These estimates are obtained using the logarithmic transform of
the concentration. The heterogeneity factor (Finney 1971, pages 70–72)
is not used. For a test material to pass the toxicity test, according to
the requirements stated in the offshore oil and gas extraction industry
BAT effluent limitations and NSPS, the LC50, adjusted for natural
responsiveness, must be greater than 3% suspended particulate phase
(SPP) concentration by volume unadjusted for the 1 to 9 dilution. Other
toxicity test models may be used to obtain toxicity estimates provided
the modeled mathematical expression for the lethality rate must increase
continuously with concentration. The lethality rate is modeled to
increase with concentration to reflect an assumed increase in toxicity
with concentration even though the observed lethality may not increase
uniformly because of the unpredictable animal response fluctuations.

The range finding test is used to establish a reasonable set of test
concentrations in order to run the definitive test. However, if the
lethality rate changes rapidly over a narrow range of concentrations,
the range finding assay may be too coarse to establish an adequate set
of test concentrations for a definitive test.

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   REF _Ref238923671 \h  Table 3-2  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% SPP with 95% limits of 3.0
and 3.5% SPP with the 1 to 9 dilution. The estimated spontaneous
mortality rate based on all of the data is 9.6%.

Table   STYLEREF 1 \s  3 -  SEQ Table \* ARABIC \s 1  2 . Listing of
Acute Toxicity Test Data (August 1983 to September 1983) With Eight
Generic Drilling Fluids and Mysid Shrimp

[fluid N2=1]

Percent Concentration	Number Exposed	Number Dead (96 hours)	Number Alive
(96 hours)

0	60	3	57

1	60	11	49

2	60	11	49

3	60	25	35

4	60	48	12

5	60	60	0

The Partial Toxicity Test for Evaluation of Test Material

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 LC50 adjusted for
natural response.

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% 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.

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 X0 of   REF _Ref238923500 \h  Table
3-3 . 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% test concentration
is less than or equal to corresponding number in the column labeled X1
then the test material passes the partial toxicity test. Otherwise the
test material fails the toxicity test.

Data shall be reported as percent suspended particulate phase.

Table   STYLEREF 1 \s  3 -  SEQ Table \* ARABIC \s 1  3 . Partial
Toxicity Test Passing Criteria

 Number Killed in Control (X0)	Number Killed in Test (X1)

0	22

1	22

2	23

3	23

4	24

5	24

6	25

References

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.

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.

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

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.

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

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

Procedure for Mixing Base Fluids With Sediments (EPA Method 1646)

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
2,000 micron mesh sieve to remove large debris. Then press-sieve 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 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.

Determining the Wet to Dry Ratio for the Control Sediment

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. Re-weigh the samples to determine the dry weight.
Determine the wet/dry ratio by dividing the net wet weight by the net
dry weight:

 	[  STYLEREF 1 \s  4 -  SEQ Equation \* ARABIC \s 1  1 ]

Determining the Density of the Wet Control or Dilution Sediment

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.

 	[  STYLEREF 1 \s  4 -  SEQ Equation \* ARABIC \s 1  2 ]

Determining the Amount of Base Fluid Needed

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:

 	[  STYLEREF 1 \s  4 -  SEQ Equation \* ARABIC \s 1  3 ]

Determine the amount of dry sediment in kilograms (kg) required for each
concentration:

 	[  STYLEREF 1 \s  4 -  SEQ Equation \* ARABIC \s 1  4 ]

Finally, determine the amount of base fluid required to spike the
control sediment at each concentration:

 	[  STYLEREF 1 \s  4 -  SEQ Equation \* ARABIC \s 1  5 ]

For spiking test substances other than pure base fluids (e.g., whole mud
formulations), determine the spike amount as follows:

 	[  STYLEREF 1 \s  4 -  SEQ Equation \* ARABIC \s 1  6 ]

Primary Mixing

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 LC50 values.

Testing for Homogeneity of Base Fluid

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.

Commencing the Sediment Toxicity Test

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 LC50,
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.

U.S. EPA. 2001. Effluent Limitations Guidelines and New Source
Performance Standards for the Oil and Gas Extraction Point Source
Category. Federal Register, 66: 6849 (22 January 2001).

U.S. EPA. 2001. Effluent Limitations Guidelines and New Source
Performance Standards for the Oil and Gas Extraction Point Source
Category: Correction. Federal Register, 66: 30811 (8 June 2001).

Protocol for the Determination of Degradation of Non-Aqueous Base
Fluids in a Marine Closed Bottle Biodegradation Test System: Modified
ISO 11734:1995 (EPA Method 1647)

Summary of Method

This method determines the anaerobic degradation potential of mineral
oils, paraffin oils and non aqueous fluids (NAF) in sediments. These
substrates are base fluids for formulating offshore drilling fluids. The
test evaluates base fluid biodegradation rates by monitoring gas
production due to microbial degradation of the test fluid in natural
marine sediment.

The test procedure places a mixture of marine/estuarine sediment, test
substrate (hydrocarbon or controls) and seawater into clean 120 ml (150
ml actual volume) Wheaton serum bottles. The test is run using four
replicate serum bottles containing 2,000 mg carbon/kg dry weight
concentration of test substrate in sediment. The use of resazurin dye
solution (1 ppm) evaluates the anaerobic (redox) condition of the
bottles (dye is blue when oxygen is present, reddish in low oxygen
conditions and colorless if oxygen free). After capping the bottles, a
nitrogen sparge removes air in the headspace before incubation begins.
During the incubation period, the sample should be kept at a constant
temperature of 29 ±1°C. Gas production and composition is measured
approximately every two weeks. The samples need to be brought to ambient
temperature before making the measurements. Measure gas production using
a pressure gauge. Barometric pressure is measured at the time of testing
to make necessary volume adjustments.

ISO 11734:1995 specifies that total gas is the standard measure of
biodegradation. While modifying this test for evaluating biodegradation
of NAFs, methane was also monitored and found to be an acceptable method
of evaluating biodegradation. Section   REF _Ref239169572 \r \h  5.7
contains the procedures used to follow biodegradation by methane
production. Measurement of either total gas or methane production is
permitted. If methane is followed, determine the composition of the gas
by using gas chromatography (GC) analysis at each sampling. At the end
of the test when gas production stops, or at around 275 days, an
analysis of sediment for substrate content is possible. Common methods
which have been successfully used for analyzing NAFs from sediments are
listed in Section   REF _Ref239153421 \r \h  5.8 .

System Requirements

This environmental test system has three phases, spiked sediment,
overlying seawater, and a gas headspace. The sediment/test compound
mixture is combined with synthetic sea water and transferred into 120 mL
serum bottles. The total volume of sediment/sea water mixture in the
bottles is 75 mL. The volume of the sediment layer will be approximately
50 mL, but the exact volume of the sediment will depend on sediment
characteristics (wet:dry ratio and density). The amount of synthetic sea
water will be calculated to bring the total volume in the bottles to 75
mL. The test systems are maintained at a temperature of 29 ±1oC during
incubation. The test systems are brought to ambient temperatures prior
to measuring pressure or gas volume.

Sample Requirements

The concentration of base fluids are at least 2,000 mg carbon test
material/kg dry sediment. Carbon concentration is determined by
theoretical composition based on the chemical formula or by chemical
analysis by ASTM D5291-96. Sediments with positive, intermediate and
negative control substances as well as a C1618 Internal Olefin type base
fluid will be run in conjunction with test materials under the same
conditions. The positive control is ethyl oleate (CAS 111-62-6), the
intermediate control is 1-hexadecene (CAS 629-73-2), and the negative
control is squalane (CAS 111-01-3). Controls must be of analytical grade
or the highest grade available. Each test control concentration should
be prepared according to the mixing procedure described in Section   REF
_Ref238986273 \r \h  \* MERGEFORMAT  5.3.1 .

Product names will be used for examples or clarification in the
following text. Any use of trade or product names in this publication is
for descriptive use only, and dos not constitute endorsement by EPA or
the authors

Seawater Requirements

Synthetic seawater at a salinity of 25 ±1 ppt should be used for the
test. The synthetic seawater should be prepared by mixing a commercially
available artificial seawater mix, into high purity distilled or
de-ionized water. The seawater should be aerated and allowed to age for
approximately one month prior to use.

Sediment Requirements

The dilution sediment must be from a natural estuarine or marine
environment and be free of the compounds of interest. The collection
location, date and time will be documented and reported. The sediment is
prepared by press-sieving through a 2,000-micron mesh sieve to remove
large debris, then press-sieving through a 500-micron sieve to remove
indigenous organisms that may confound test results. The water content
of the sediment should be less than 60% (w/w) or a wet to dry ratio of
2.5. The sediment should have a minimum organic matter content of 3%
(w/w) as determined by ASTM D2974-87 (95) (Method A and D and calculate
organic matter as in Section 12 of method ASTM D2974-87).

To reduce the osmotic shock to the microorganisms in the sediment the
salinity of the sediment’s pore water should be between 20-30 ppt.
Sediment should be used for testing as soon as possible after field
collection. If required, sediment can be stored in the dark at 4°C with
3-6 inches of overlying water in a sealed container for a maximum period
of 2 months prior to use.

Test Set Up

The test is set up by first mixing the test  or control substrates into
the sediment inoculum, then mixing in seawater to make a pourable
slurry. The slurry is then poured into serum bottles, which are then
flushed with nitrogen and sealed.

Mixing Procedure

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

Determine the wet to dry weight ratio for the control sediment by
weighing approximately 10 sub-samples of approximately 1 g each of the
screened and homogenized wet sediment into tared aluminum weigh pans.
Dry sediment at 105°C for18-24 h. Remove the dried sediments and cool
in a desiccator. Repeat the drying, cooling, and weighing cycle until a
constant weight is achieved (within 4% of previous weight). Re-weigh the
samples to determine the dry weight. Calculate the mean wet and dry
weights of the 10 sub samples and determine the wet/dry ratio by
dividing the mean wet weight by the mean dry weight using Equation 5-1.
This is required to determine the weight of wet sediment needed to
prepare the test samples.

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  1 ]

Determine the density (g/ml) of the wet sediment. This will be used to
determine total volume of wet sediment needed for the various test
treatments. One method is to tare a 5 ml graduated cylinder and add
about 5 ml of homogenized sediment. Carefully record the volume then
weigh this volume of sediment. Repeat this a total of three times. To
determine the wet sediment density, divide the weight by volume per the
following formula:

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  2 ]

Determine the amount of base fluid to be spiked into wet sediment in
order to obtain the desired initial base fluid concentration of 2,000 mg
carbon/kg dry weight. An amount of wet sediment that is the equivalent
of 30 g of dry sediment will be added to each bottle. A typical
procedure is to prepare enough sediment for 8 serum bottles (3 bottles
to be sacrificed at the start of the test, 4 bottles incubated for
headspace analysis, and enough extra sediment for 2 extra  bottles).
Extra sediment is needed  because some of the sediment will remain
coated onto the mixing bowl and utensils. Experience with this test may
indicate that preparing larger volumes of spiked sediment is a useful
practice, then the following calculations should be adjusted
accordingly.

Determine the total weight of dry sediment needed to add 30 g dry
sediment to 8 bottles. If more bottles are used then the calculations
should be modified accordingly. For example:

	30 g dry sediment per bottle × 8 = 240 g dry sediment	[  STYLEREF 1 \s
 5 -  SEQ Equation \* ARABIC \s 1  3 ]

Determine the weight of base fluid, in terms of carbon, needed to obtain
a final base fluid concentration of 2,000 mg carbon/kg dry weight. For
example:

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  4 ]

Convert from mg of carbon to mg of base fluid. This calculation will
depend on the % fraction of carbon present in the molecular structure of
each base fluid. For the control fluids, ethyl oleate is composed of
77.3% carbon, hexadecene is composed of 85.7% carbon, and squalane is
composed of 85.3% carbon. The carbon fraction of each base fluid should
be supplied by the manufacturer or determined before use. ASTM D5291-96
or equivalent will used to determine composition of fluid.

To calculate the amount of base fluid to add to the sediment, divide the
amount of carbon  (480 mg) by the percent fraction of carbon in the
fluid.

For example, the amount of ethyl oleate added to 240 g dry weight
sediment can be calculated from the following equation:

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  5 ]

Therefore, add 621 mg of ethyl oleate to 240 g dry weight sediment for a
final concentration of 2,000 mg carbon/kg sediment dry weight.

Mix the calculated amount of base fluid with the appropriate weight of
wet sediment.

Use the wet:dry ratio to convert from g sediment dry weight to g
sediment wet weight, as follows:

	240 g dry sediment ×wet:dry ratio = g wet sediment needed	[  STYLEREF
1 \s  5 -  SEQ Equation \* ARABIC \s 1  6 ]

Weigh the appropriate amount of base fluid (calculated in Section   REF
_Ref239152809 \r \h  \* MERGEFORMAT  <IP_ADDRESS>c ) into stainless mixing
bowls, tare the vessel weight, then add the wet sediment calculated in
Equation 5-5, and mix with a high shear dispersing impeller for 9
minutes.

The sediment is now mixed with synthetic sea water to form a slurry that
will be transferred into the bottles.

Creating Seawater/Sediment Slurry

Given that the total volume of sediment/sea water slurry in each bottle
is to be 75 mL, determine the volume of sea water to add to the wet
sediment.

If each bottle is to contain 30 g dry sediment, calculate the weight,
and then the volume, of wet sediment to be added to each bottle

	30 g dry sediment × wet:dry ratio = g wet sediment added to each
bottle	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  7 ]

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  8 ]

Calculate volume of sea water to be added to each bottle

	75 mL total volume – mL wet sediment (from Eq. 8) = mL of sea water	[
 STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  9 ]

Determine the ratio of sea water to wet sediment (volume:volume) in each
bottle

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  10 ]

Convert the wet sediment weight from Equation 5-6 into a volume using
the sediment density.

	g wet sediment (Eq. 5-6) density = volume (mL) of sediment	[  STYLEREF
1 \s  5 -  SEQ Equation \* ARABIC \s 1  11 ]

Determine the amount of sea water to mix with the wet sediment.

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  12 ]

Mix sea water thoroughly with wet sediment to form a sediment/sea water
slurry.

Bottling the Sediment Seawater Slurry

The total volume of sediment/sea water slurry in each bottle is to be 75
mL. Convert the volume (mL) of sediment/sea water slurry into a weight
(g) using the density of the sediment and the seawater.

Determine the weight of sediment to be added to each bottle

	mL sediment (Eq. 5-8) × density of wet sediment (g/mL) = g wet
sediment	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  13 ]

Determine the weight of sea water to be added to each bottle

	mL sea water (Eq. 5-9) × density of sea water (1.01 g/mL) = g sea
water	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  14 ]

Determine weight of sediment/sea water slurry to be added to each bottle

	g wet sediment (Eq. 5-13) + g sea water (Eq. 5-14) = g sediment/sea
water slurry	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  15 ]

This should provide each bottle with 30 g dry sediment in a total volume
of 75 mL.

Putting the sediment:seawater slurry in the serum bottles.

Note: The slurry will need to be constantly stirred to keep the sediment
suspended.

μL) of a 1 gram/L resazurin dye stock solution. Cap the bottle with a
butyl rubber stopper (Bellco Glass, Part #2048- 11800) and crimp with an
aluminum seal (Bellco Glass Part #2048-11020).

Using a plastic tube with a (23 gauge, 1 inch long) needle attached to
one side and a nitrogen source to the other, puncture the serum cap with
the needle. Puncture the serum cap again with a second needle to sparge
the bottle’s headspace of residual air for two minutes. The nitrogen
should be flowing at no more than 100 mL/min to encourage gentle
displacement of oxygenated air with nitrogen. Faster nitrogen flow rates
would cause mixing and complete oxygen removal would take much longer.
Remove the nitrogen needle first to avoid any initial pressure problems.
The second (vent) needle should be removed within 30 seconds of removing
the nitrogen needle.

Triplicate blank test systems are prepared, with similar quantities of
sediment and seawater without any base fluid. Incubate in the dark at a
constant temperature of 29 1°C.

Record the test temperature. The test duration is dependent on base
fluid performance, but at a maximum should be no more than 275 days.
Stop the test after all base fluids have achieved a plateau of gas
production. At termination, base fluid concentrations can be verified in
the terminated samples by extraction and GC analysis according to
Section   REF _Ref239153911 \r \h  5.8 .

Concentration Verification Chemical Analyses

C and a sample of sediment from each bottle should be analyzed for
base fluid content as soon as possible. The coefficient of variation
(CV) for the replicate samples must be less than 20%. The results should
show recovery of at least 70% of the spiked base fluid. Use an
appropriate analytical procedure described in Section   REF
_Ref239153911 \r \h  5.8  to perform the extractions and analyses. If
any set of sediments fail the criteria for concentration verification,
then the corrective action for that set of sediments is also outlined in
Section   REF _Ref239153911 \r \h  5.8 .

The nominal concentrations and the measured concentrations from the
three bottles selected for concentration verification should be reported
for the initial test concentrations. 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 must be stopped and
restarted with adequately mixed sediment.

Gas Monitoring Procedures

Biodegradation is measured by total gas as specified in ISO 11734:1995.
Methane production can also

be tracked and is described in Section   REF _Ref239153759 \r \h  5.7 .

Total Gas Monitoring Procedures

Bottles should be brought to room temperature before readings are taken.
The bottles are observed to confirm that the resazurin has not oxidized
to pink or blue. Total gas production in the culture bottles should be
measured using a pressure transducer (one source is Biotech
International). The pressure readings from test and control cultures are
evaluated against a calibration curve created by analyzing the pressure
created by known additions of gas to bottles established identically to
the culture bottles. Bottles used for the standard curve contain 75 mL
of water, and are sealed with the same rubber septa and crimp cap seals
used for the bottles containing sediment. After the bottles used in the
standard curve have been sealed, a syringe needle inserted through the
septa is used to equilibrate the pressure inside the bottles to the
outside atmosphere. The syringe needle is removed and known volumes of
air are injected into the headspace of the bottles. Pressure readings
provide a standard curve relating the volume of gas injected into the
bottles and headspace pressure. No less than three points may be used to
generate the standard curve. A typical standard curve may use 0, 1, 5,
10, 20 and 40 ml of gas added to the standard curve bottles.

The room temperature and barometric pressure (to two digits) should be
recorded at the time of sampling. One option for the barometer is Fisher
Part #02-400 or 02-401. Gas production by the sediment is expressed in
terms of the volume (mL) of gas at standard temperature (0°C = 273°K)
and pressure (1 atm = 30 inches of Hg) using Eq. 5-16.

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  16 ]

Where:

V2	=	Volume of gas production at standard temperature and pressure

P1	=	Barometric pressure on day of sampling (inches of Hg)

V1 	=	Volume of gas measured on day of sampling (mL)

T2 	=	Standard temperature = 273°K

T1 	=	Temperature on day of sampling (°C + 273 = °K)

P2 	=	Standard pressure = 30 inches Hg

An estimate can be made of the total volume of anaerobic gas that will
be produced in the bottles. The gas production measured for each base
fluid can be expressed as a percent of predicted total anaerobic gas
production.

Calculate the total amount of carbon in the form of the base fluid
present in each bottle

Each bottle is to contain 30 g dry weight sediment. The base fluid
concentration is 2,000 mg carbon/kg dry weight sediment. Therefore:

	2,000 mg carbon/kg sediment × (30 g ÷ 1,000) = 60 mg carbon per
bottle	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  17 ]

Theory states that anaerobic microorganisms will convert 1 mole of
carbon substrate into 1 mole of total anaerobic gas production

Calculate the number of moles of carbon in each bottle.

The molecular weight of carbon is 12 (i.e., 1 mole of carbon = 12 g).
Therefore, the number of moles of carbon in each bottle can be
calculated.

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  18 ]

Calculate the predicted volume of anaerobic gas

One mole of gas equals 22.4 L (at standard temperature and pressure),
therefore,

	0.005 moles × 22.4 L = 0.112L (or 112 mL total gas production)	[
STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  19 ]

Gas Venting

If the pressure in the serum bottle is too great for the pressure
transducer or syringe, some of the excess gas must be wasted. The best
method to do this is to vent the excess gas right after measurement. To
do this, remove the barrel from a 10-mL syringe and fill it 1/3 full
with water. This is then inserted into the bottle through the stopper
using a small diameter (high gauge) needle. The excess pressure is
allowed to vent through the water until the bubbles stop. This allows
equalization of the pressure inside the bottle to atmospheric without
introducing oxygen. The amount of gas vented (which is equal to the
volume determined that day) must be kept track of each time the bottles
are vented. A simple way to do this in a spreadsheet format is to have a
separate column in which cumulative vented gas is tabulated. Each time
the volume of gas in the cultures is analyzed, the total gas produced is
equal to the gas in the culture at that time plus the total of the
vented gas.

To keep track of the methane lost in the venting procedure, multiply the
amount of gas vented each time by the corrected % methane determined on
that day. The answer gives the volume of methane wasted. This must be
added into the cumulative totals similarly to the total gas additions.

Test Acceptability and Interpretation

Test Acceptability

At day 275 or when gas production has plateaued, whichever is first, the
controls are evaluated to confirm that the test has been performed
appropriately. In order for this modification of the closed bottle
biodegradation test to be considered acceptable, all the controls must
meet the biodegradation levels indicated in   REF _Ref238923696 \h
Table 5-1 . The intermediate control hexadecene must produce at least
30% of the theoretical gas production. This level may be reexamined
after two years and more data has been generated.

Table   STYLEREF 1 \s  5 -  SEQ Table \* ARABIC \s 1  1 . Test
Acceptability Criteria

Concentration	Percent Biodegradability as a Function of Gas Measurement

	Positive control	Squalane negative control	Hexadecene intermediate
control

2,000 mg carbon/kg	≥60% theoretical	≤5% theoretical	≥30%
theoretical

Interpretation

In order for a fluid to pass the closed bottle test, the biodegradation
of the base fluid as indicated by the total amount of total gas (or
methane) generated once gas production has plateaued (or at the end of
275 days, which ever is first) must be greater than or equal to the
volume of gas (or methane) produced by the reference standard (internal
elefin or ester).

The method for evaluating the data to determine whether a fluid has
passed the biodegradation test must use the equations:

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  20 ]

Where:

NAF		=	Stock base fluid being tested for compliance

Reference fluid	=	C16-C18 internal olefin or C12-C14 or C8 ester
reference

			fluid

Methane Measurement

Methane Monitoring Procedures

The use of total gas production alone may result in an underestimation
of the actual metabolism occurring since CO2 is slightly soluble in
water. An acceptable alternative method is to monitor methane production
and total gas production. This is easily done using GC analysis. A
direct injection of headspace gases can be made into a GC using almost
any packed or capillary column with an FID detector. Unless volatile
fuels or solvents are present in the test material or the inocula, the
only component of the headspace gas that can be detected using an FID
detector is methane. The percent methane in the headspace gas is
determined by comparing the response of the sample injections to the
response from injections of known percent methane standards. The percent
methane is corrected for water vapor saturation using Eq. 5-21 and then
converted to a volume of dry methane using Eq. 5-22.

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  21 ]

Where:

D	=	The density of water vapor at saturation (g/m3, can be found in CRC
Handbook of Chemistry and Physics) for the temperature of sampling.

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  22 ]

Where:

VCH4 	=	The volume of methane in the bottle

S 	= 	Volume of excess gas production (measured with a pressure
transducer)

V 	=	Volume of the headspace in the culture bottle (total volume -
liquid phase)

P 	= 	Barometric pressure (mm Hg, measured with barometer)

T 	= 	Temperature (C)

Pw 	= 	Vapor pressure of water at T (mm Hg, can be found in CRC Handbook
of Chemistry and Physics)

CH4 	= 	% methane in headspace gas (after correction for water vapor)

The total volume of serum bottles sold as 125 mL bottles (Wheaton) is
154.8 mL.

The volumes of methane produced are then compared to the volumes of
methane in the controls to determine if a significant inhibition of
methane production or a significant increase of methane production has
been observed. Effective statistical analyses are important, as
variability in the results is common due to the heterogeneity of the
inoculum’s source. It is also common to observe that the timing of the
initiation of culture activity is not equal in all of the cultures.
Expect a great variability over the period when the cultures are active,
some replicates will start sooner than others, but all of the replicates
should eventually reach similar levels of base fluid degradation and
methane production.

Expected Methane Production Calculations

The amount of methane expected can be calculated using the equation of
Symons and Buswell (Eq. 5-23). In the case of complete mineralization,
all of the carbon will appear as wither CO2 or CH4, thus the total moles
of gas produced will be equal to the total moles of carbon in the parent
molecule. The use of the Buswell equation allows you to calculate the
effects the redox potential will have on the distribution of the
products in methanogenic cultures. More reduced electron donors will
allow the production of more methane, while more oxidized electron
donors will cause a production of more carbon dioxide.

	CnHaObNcSd + (n-a/4 -b/2 + 7c/4 + d/2) H2O → (n/2 -a/8+b/4-5c/8 +
d/4) CO2 +

	(n/2 +a/8 -b/4 -3c/8-d/4) CH4 + cNH4HCO3 + dH2S	[  STYLEREF 1 \s  5 -
SEQ Equation \* ARABIC \s 1  23 ]

An example calculation of the expected methane volume in a culture fed
2,000 mg/kg hexadecene is as follows. The application of Symons and
Buswell’s equation reveals that hexadecene (C16H32) will yield 4 moles
of CO2 and 12 moles of CH4. Assuming 30 g of dry sediment are added to
the bottles with 2,334 mg hexadecene/kg dry sediment (i.e., equivalent
to 2,000 mg carbon/kg dry sediment) the calculation is as follows.

 	[  STYLEREF 1 \s  5 -  SEQ Equation \* ARABIC \s 1  24 ]

By subtracting the average amount of methane in control bottles from the
test bottles and then dividing by the expected volume an evaluation of
the completion of the process may be conducted.

Concentration Verification Analysis

≤20% Coefficient of Variability and an average of ≥70% to ≤120% of
fluid delivered to sediment.

If a third party performs the analysis, then the laboratory should be
capable of delivering the homogeneity data within seven days, in order
to identify any samples that do not meet the homogeneity requirement as
quickly as possible.

If one sediment/fluid set, out a multiple set batch of samples, fails
these criteria, then that one set of samples must be discarded and a
fresh set of spiked sediment prepared, started, and analyzed to ensure
homogeneity. The same stock sediment is used to prepare the replacement
set(s). The remaining sets do not need to be re-mixed or restarted.

The re-mixed set(s) will need to be run the additional days as
appropriate to ensure that the total number of days is the same for all
sets of bottles, even though the specific days are not aligned.

Re-mixing of bottle sets can be performed multiple times as a result of
a failure of the analytical criteria, until the holding time for the
stock sediment has expired (60 days). If the problem set(s) has not
fallen within the acceptable analytical criteria by then, it must not be
part of the batch of bottles run. If the problem batch is one of the
controls, and those controls were not successfully prepared when the
sediment holding time expired, then the entire test must be restarted.

Program Quality Assurance and Quality Control

Calibration

All equipment / instrumentation will be calibrated in accordance with
the test method or the manufacture's instructions and may be scheduled
or triggered.

Where possible, standards used in calibration will be traceable to a
nationally recognized standard (e.g., certified standard by NIST).

All calibration activities will be documented and the records retained.

The source, lot, batch number, and expiration date of all reagents used
with be documented and retained.

Maintenance

All equipment / instrumentation will be maintained in accordance with
the test method or the manufacture's instructions and may be scheduled
or triggered.

All maintenance activities will be documented and the records retained.

Data Management and Handling

All primary (raw) data will be correct, complete, without selective
reporting, and will be maintained.

Hand-written data will be recorded in lab notebooks or electronically at
the time of observation.

All hand-written records will be legible and amenable to reproduction by
electrostatic copiers.

All changes to data or other records will be made by:

Using a single line to mark-through the erroneous entry (maintaining
original data legibility)

Write the revision

Initial, date, and provide revision code (see attached or laboratory’s
equivalent)

All data entry, transcriptions, and calculations will be verified by a
qualified person.

Verification will be documented by initials of verifier and date

Procedures will be in place to address data management procedures used
(at minimum):

Significant figures

Rounding practices

Identification of outliers in data series

Required statistics

Document Control

All technical procedures, methods, work instructions, standard operating
procedures must be documented and approved by laboratory management
prior to the implementation.

All primary data will be maintained by the contractor for a minimum of
five (5) years.

Personnel and Training

Only qualified personnel shall perform laboratory activities.

Records of staff training and experience will be available. This will
include initial and refresher training (as appropriate).

Test Performance

All testing will done in accordance with the specified test methods.

Receipt, arrival condition, storage conditions, dispersal, and
accountability of the test article will be documented and maintained.

Receipt or production, arrival or initial condition, storage conditions,
dispersal, and accountability of the test matrix (e.g., sediment or
artificial seawater) will be documented and maintained.

Source, receipt, arrival condition, storage conditions, dispersal, and
accountability of the test organisms (including inoculum) will be
documented and maintained.

Actual concentrations administered at each treatment level will be
verified by appropriate methodologies.

Any data originating at a different laboratory will be identified and
the laboratory fully referenced in the final report.

The following references identify analytical methods that have
historically been successful for achieving the analytical quality
criteria.

Continental Shelf Associates report 1998. Joint EPA/Industry screening
survey to assess the deposition of drill cuttings and associated
synthetic based mud on the seabed of the Louisiana continental shelf,
Gulf of Mexico. Analysis by Charlie Henry report Number IES/RCAT97-36
GC-FID and GC/MS.

EPA Method 3550 for extraction with EPA Method 8015 for GC-FID.

Webster, L; Mackie, P.R.; Hird, S.J.; Munro, P.D.; Brown, N.A. and
Moffatt, C.F. (1997) Development of Analytical Methods for the
Determination of Synthetic Mud Base Fluids in Marine Sediments Analyst
122:1485-1490.

Munro, P.D., B Croce, C.F. Moffet, N.A Brown, A.D. McIntosh, S.J. Hird,
R.M. Stagg. 1998. Solid-phase test for comparison for degradation rates
of synthetic mud base fluids used in the off shore drilling industry.
Environ. Toxicol. Chem. 17:1951-1959.

Determination of Crude Oil Contamination in Non-Aqueous Drilling Fluids
by Gas Chromatography/Mass Spectrometry (GC/MS) (EPA Method 1655)

Scope and Application

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.

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

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.

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   REF
_Ref239492653 \r \h  6.7 ,   REF _Ref239038963 \r \h  6.9.2 , and   REF
_Ref239042712 \r \h  6.12 .

Summary of Method

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.

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

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.

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.

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.

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.

Quality is assured through reproducible calibration and testing of GC/MS
system and through analysis of quality control samples.

Definitions

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).

TIC—Total Ion Chromatograph.

EIP—Extracted Ion Profile.

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

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

Interferences and Limitations

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

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.

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

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

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.

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.

Safety

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.

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.

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 Section   REF _Ref239157123 \r \h  6.16 , References 1
through 3.

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

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.

Equipment for glassware cleaning.

Laboratory sink with overhead fume hood.

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).

Equipment for sample preparation.

Laboratory fume hood.

Analytical balance—Capable of weighing 0.1 mg.

Glassware.

Disposable pipettes—Pasteur, 150 mm long by 5 mm ID (Fisher Scientific
13–678–6A, or equivalent) baked at 400°C for a minimum of 1 hour.

Glass volumetric pipettes or gas tight syringes—1.0-mL ±1% and 0.5-mL
±1%.

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

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

Centrifuge and centrifuge tubes—Centrifuge capable of 10,000 rpm, or
better, (International Equipment Co., IEC Centra MP4 or equivalent) and
50-mL centrifuge tubes (Nalgene, Ultratube, Thin Wall 25×89 mm,
#3410–2539).

Gas Chromatograph/Mass Spectrometer (GC/MS):

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

Column—30 m (or 60 m) × 0.32 mm ID (or 0.25 mm ID) 1 µm film
thickness (or 0.25 µm film thickness) silicone-coated fused-silica
capillary column (J&W Scientific DB–5 or equivalent).

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).

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

Data 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.

Reagents and Standards

Methylene chloride—Pesticide grade or equivalent. Use when necessary
for sample dilution.

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.

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

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

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.

GC/MS system performance test mix (SPTM) standards—The SPTM standards
shall contain octane, decane, dodecane, tetradecane, tetradecene,
toluene, ethylbenzene, 1,2,4-trimethylbenzene, 1-methylnaphthalene and
1,3-dimethylnaphthalene. 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.

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 section using the Reference Crude Oil:

Label 4 jars with the following identification: Jar 1—0%Ref-IOLab, Jar
2—0.5%Ref-IOLab, Jar 3—1%Ref-IOLab, and Jar 4—2%Ref-IOLab.

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

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.

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

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 µL of the 0.01g/mL
1,3,5-trichlorobenzene internal standard solution (see Section   REF
_Ref239492726 \r \h  <IP_ADDRESS> ), cap with a Teflon lined crimp cap, and
vortex for ca. 10 sec.

Repeat step <IP_ADDRESS>(e) except use an aliquot from Jar 2.

Repeat step <IP_ADDRESS>(e) except use an aliquot from Jar 3.

Repeat step <IP_ADDRESS>(e) except use an aliquot from Jar 4.

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

Precision and recovery standard (mid level crude oil/drilling fluid
calibration standard)—Prepare a mid point crude oil/ drilling fluid
calibration using IO Lab drilling fluid and Reference Oil at a
concentration of 1.0% by weight. Prepare this standard according to the
procedures outlined in Section   REF _Ref239492767 \r \h  <IP_ADDRESS>a
through   REF _Ref239041252 \r \h  <IP_ADDRESS>e , with the exception that
only “Jar 3” needs to be prepared. Remove and spike with internal
standard, as many 0.5-g aliquots as needed to complete the GC/MS
analysis (see Section   REF _Ref239041577 \r \h  6.11.6 —bracketing
authentic samples every 12 hours with precision and recovery standard)
and the initial demonstration exercise described in Section   REF
_Ref239038963 \r \h  6.9.2 .

Stability of standards

When not used, standards shall be stored in the dark, at -5 to -20°C in
screw-capped vials with PTFE-lined lids. Place a mark on the vial at the
level of the solution so that solvent loss by evaporation can be
detected. Bring the vial to room temperature prior to use.

Solutions used for quantitative purposes shall be analyzed within 48
hours of preparation and on a monthly basis thereafter for signs of
degradation. A standard shall remain acceptable if the peak area remains
within ±15% of the area obtained in the initial analysis of the
standard.

Sample Collection Preservation and Storage

Collect NAF and base fluid samples in 100- to 200-mL glass bottles with
PTFE- or aluminum foil lined caps.

Samples collected in the field shall be stored refrigerated until time
of preparation.

Sample and extract holding times for this method have not yet been
established. However, based on initial experience with the method,
samples should be analyzed within seven to ten days of collection and
extracts should be analyzed within seven days of preparation.

After completion of GC/MS analysis, extracts shall be refrigerated at
4°C until further notification of sample disposal.

Quality Control

Each laboratory that uses this method is required to operate a formal
quality assurance program (Section   REF _Ref239041902 \r \h  6.16 ,
Reference 4). The minimum requirements of this program shall consist of
an initial demonstration of laboratory capability, and ongoing analysis
of standards, and blanks as a test of continued performance, analyses of
spiked samples to assess accuracy and analysis of duplicates to assess
precision. Laboratory performance shall be compared to established
performance criteria to determine if the results of analyses meet the
performance characteristics of the method.

The analyst shall make an initial demonstration of the ability to
generate acceptable accuracy and precision with this method. This
ability shall be established as described in Section   REF _Ref239038963
\r \h  \* MERGEFORMAT  6.9.2 .

The analyst is permitted to modify this method to improve separations or
lower the cost of measurements, provided all performance requirements
are met. Each time a modification is made to the method, the analyst is
required to repeat the calibration (Section   REF _Ref239042041 \r \h
\* MERGEFORMAT  6.10.4 ) and to repeat the initial demonstration
procedure described in Section   REF _Ref239038963 \r \h  \* MERGEFORMAT
 6.9.2 .

Analyses of blanks are required to demonstrate freedom from
contamination. The procedures and criteria for analysis of a blank are
described in Section   REF _Ref239042114 \r \h  \* MERGEFORMAT  6.9.3 .

Analysis of a matrix spike sample is required to demonstrate method
accuracy. The procedure and QC criteria for spiking are described in
Section   REF _Ref239042138 \r \h  \* MERGEFORMAT  6.9.4 .

Analysis of a duplicate field sample is required to demonstrate method
precision. The procedure and QC criteria for duplicates are described in
Section   REF _Ref239042160 \r \h  \* MERGEFORMAT  6.9.5 .

Analysis of a sample of the clean NAF(s) (as sent from the
supplier—has not been circulated downhole) used in the drilling
operations is required.

The laboratory shall, on an ongoing basis, demonstrate through
calibration verification and the analysis of the precision and recovery
standard (Section   REF _Ref239042200 \r \h  \* MERGEFORMAT  <IP_ADDRESS>  )
that the analysis system is in control. These procedures are described
in Section   REF _Ref239041577 \r \h  \* MERGEFORMAT  6.11.6 .

The laboratory shall maintain records to define the quality of data that
is generated.

Initial precision and accuracy—The initial precision and recovery test
shall be performed using the precision and recovery standard (1% by
weight Reference Oil in IO Lab drilling fluid). The laboratory shall
generate acceptable precision and recovery by performing the following
operations.

Prepare four separate aliquots of the precision and recovery standard
using the procedure outlined in Section   REF _Ref239042200 \r \h  \*
MERGEFORMAT  <IP_ADDRESS>  . Analyze these aliquots using the procedures
outlined in Section   REF _Ref239042570 \r \h  \* MERGEFORMAT  6.11 .

Using the results of the set of four analyses, compute the average
recovery (X) in weight percent and the standard deviation of the
recovery(s) for each sample.

If s and X meet the acceptance criteria of 80% to 110%, system
performance is acceptable and analysis of samples may begin. If,
however, s exceeds the precision limit or X falls outside the range for
accuracy, system performance is unacceptable. In this event, review this
method, correct the problem, and repeat the test.

Accuracy and precision—The average percent recovery (P) and the
standard deviation of the percent recovery (Sp) Express the accuracy
assessment as a percent recovery interval from P–2Spto P+2Sp. For
example, if P=90% and Sp=10% for four analyses of crude oil in NAF, the
accuracy interval is expressed as 70% to 110%. Update the accuracy
assessment on a regular basis.