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  3.7.1.3
).

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 
3.7.1.5  through   REF _Ref239147771 \r \h  \* MERGEFORMAT  3.7.1.7 )
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  5.3.1.3c ) 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  6.7.2.3 ), cap with a Teflon lined crimp cap, and
vortex for ca. 10 sec.

Repeat step 6.7.2.5(e) except use an aliquot from Jar 2.

Repeat step 6.7.2.5(e) except use an aliquot from Jar 3.

Repeat step 6.7.2.5(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  6.7.2.5a 
through   REF _Ref239041252 \r \h  6.7.2.5e , 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  6.7.2.6  )
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  6.7.2.6  . 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.

Blanks—Rinse glassware and centrifuge tubes used in the method with 30
mL of methylene chloride, remove a 0.5-g aliquot of the solvent, spike
it with the 500 µL of the internal standard solution (Section   REF
_Ref239041418 \r \h  \* MERGEFORMAT  6.7.2.3 ) and analyze a 1-µL
aliquot of the blank sample using the procedure in Section   REF
_Ref239042689 \r \h  \* MERGEFORMAT  6.11 . Compute results per Section 
 REF _Ref239042712 \r \h  \* MERGEFORMAT  6.12 .

Matrix spike sample—Prepare a matrix spike sample according to
procedure outlined in Section   REF _Ref239042200 \r \h  \* MERGEFORMAT 
6.7.2.6 . Analyze the sample and calculate the concentration (% oil) in
the drilling fluid and % recovery of oil from the spiked drilling fluid
using the methods described in Sections   REF _Ref239042771 \r \h  \*
MERGEFORMAT  6.11  and   REF _Ref239042782 \r \h  \* MERGEFORMAT  6.12 .

Duplicates—A duplicate field sample shall be prepared and analyzed
according to Section   REF _Ref239042801 \r \h  \* MERGEFORMAT  6.11 .
The relative percent difference (RPD) of the calculated concentrations
shall be less than 15%.

Analyze each of the duplicates per the procedure in Section   REF
_Ref239043240 \r \h  \* MERGEFORMAT  6.11  and compute the results per
Section   REF _Ref239043270 \r \h  \* MERGEFORMAT  6.12 .

ng equation:

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

Where:

D1	= 	Concentration of crude oil in the sample; and

D2	= 	Concentration of crude oil in the duplicate sample.

If the RPD criteria are not met, the analytical system shall be judged
to be out of control, and the problem must be immediately identified and
corrected, and the sample batch re-analyzed.

A clean NAF sample shall be prepared and analyzed according to Section
6.11. Ultimately the oil-equivalent concentration from the TIC or EIP
signal measured in the clean NAF sample shall be subtracted from the
corresponding authentic field samples in order to calculate the true
contaminant concentration (% oil) in the field samples (see Section  
REF _Ref239043745 \r \h  \* MERGEFORMAT  6.12 ).

The specifications contained in this method can be met if the apparatus
used is calibrated properly, and maintained in a calibrated state. The
standards used for initial precision and recovery (Section   REF
_Ref239038963 \r \h  \* MERGEFORMAT  6.9.2  ) and ongoing precision and
recovery (Section   REF _Ref239041577 \r \h  \* MERGEFORMAT  6.11.6 )
shall be identical, so that the most precise results will be obtained.
The GC/MS instrument will provide the most reproducible results if
dedicated to the setting and conditions required for the analyses given
in this method.

Depending on specific program requirements, field replicates and field
spikes of crude oil into samples may be required when this method is
used to assess the precision and accuracy of the sampling and sample
transporting techniques.

Calibration

Establish gas chromatographic/mass spectrometer operating conditions
given in   REF _Ref238292704 \h  Table 6-1 . Perform the GC/MS system
hardware-tune as outlined by the manufacture. The gas chromatograph
shall be calibrated using the internal standard technique.

Note: Because each GC is slightly different, it may be necessary to
adjust the operating conditions (carrier gas flow rate and column
temperature and temperature program) slightly until the retention times
in   REF _Ref238292711 \h  Table 6-2  are met.

Table   STYLEREF 1 \s  6 -  SEQ Table \* ARABIC \s 1  1 . Gas
Chromatograph/Mass Spectrometer (GC/MS) Operation Conditions

Parameter	Setting

Injection port	280°C

Transfer line	280°C

Detector	280°C

Initial Temperature	50°C

Initial Time	5 minutes

Ramp	50 to 300°C @ 5°C per minute

Final Temperature	300°C

Final Hold	20 minutes or until all peaks have eluted

Carrier Gas	Helium

Flow rate	As required for standard operation

Split ratio	As required to meet performance criteria (~1:100)

Mass range	35 to 600 amu.



Table   STYLEREF 1 \s  6 -  SEQ Table \* ARABIC \s 1  2 . Approximate
Retention Time for Compounds

Compound	Approximate retention time (minutes)

Toluene	5.6

Octane, n−C8	7.2

Ethylbenzene	10.3

1,2,4-Trimethylbenzene	16.0

Decane, −C10	16.1

TCB (Internal Standard)	21.3

Dodecane, −C12	22.9

1-Methylnaphthalene	26.7

1-Tetradecene	28.4

Tetradecane, −C14	28.7

1,3-Dimethylnaphthalene	29.7



Internal standard calibration procedure—1,3,5-trichlorobenzene (TCB)
has been shown to be free of interferences from diesel and crude oils
and is a suitable internal standard.

The system performance test mix standards prepared in Section   REF
_Ref239045057 \r \h  \* MERGEFORMAT  6.7.2.4  shall be used to establish
retention times and establish qualitative detection limits.

Spike a 500-mL aliquot of the 1.25 mg/mL SPTM standard with 500 µL of
the TCB internal standard solution.

Inject 1.0 µL of this spiked SPTM standard onto the GC/MS in order to
demonstrate proper retention times. For the GC/MS used in the
development of this method the ten compounds in the mixture had typical
retention times shown in   REF _Ref238292711 \h  Table 6-2  above.
Extracted ion scans for m/z 91 and 105 showed a maximum abundance of
400,000.

Spike a 500-mL aliquot of the 0.125 mg/mL SPTM standard with 500 µL of
the TCB internal standard solution.

Inject 1.0 µL of this spiked SPTM standard onto the GC/MS to monitor
detectable levels. For the GC/MS used in the development of this test,
all ten compounds showed a minimum peak height of three times signal to
noise. Extracted ion scans for m/z 91 and 105 showed a maximum abundance
of 40,000.

GC/MS crude oil/drilling fluid calibration—There are two methods of
quantification: Total Area Integration (C8–C13) and EIP Area
Integration using m/z's 91 and 105. The Total Area Integration method
should be used as the primary technique for quantifying crude oil in
NAFs. The EIP Area Integration method should be used as a confirmatory
technique for NAFs. However, the EIP Area Integration method shall be
used as the primary method for quantifying oil in enhanced mineral oil
(EMO) based drilling fluid. Inject 1.0 µL of each of the four crude
oil/drilling fluid calibration standards prepared in Section   REF
_Ref239045212 \r \h  \* MERGEFORMAT  6.7.2.5  into the GC/MS. The
internal standard should elute approximately 21–22 minutes after
injection. For the GC/MS used in the development of this method, the
internal standard peak was (35 to 40)% of full scale at an abundance of
about 3.5e+07.

Total Area Integration Method—For each of the four calibration
standards obtain the following: Using a straight baseline integration
technique, obtain the total ion chromatogram (TIC) area from C8 to C13.
Obtain the TIC area of the internal standard (TCB). Subtract the TCB
area from the C8–C13area to obtain the true C8–C13 area. Using the
C8–C13 and TCB areas, and known internal standard concentration,
generate a linear regression calibration using the internal standard
method. The r2 value for the linear regression curve shall be greater
than or equal to 0.998. Some synthetic fluids might have peaks that
elute in the window and would interfere with the analysis. In this case
the integration window can be shifted to other areas of scan where there
are no interfering peaks from the synthetic base fluid.

EIP Area Integration—For each of the four calibration standards
generate Extracted Ion Profiles (EIPs) for m/z 91 and 105. Using
straight baseline integration techniques, obtain the following EIP
areas:

For m/z 91 integrate the area under the curve from approximately 9
minutes to 21–22 minutes, just prior to but not including the internal
standard.

For m/z 105 integrate the area under the curve from approximately 10.5
minutes to 26.5 minutes.

Obtain the internal standard area from the TCB in each of the four
calibration standards, using m/z 180.

Using the EIP areas for TCB, m/z 91 and m/z105, and the known
concentration of internal standard, generate linear regression
calibration curves for the target ions 91 and 105 using the internal
standard method. The r2 value for each of the EIP linear regression
curves shall be greater than or equal to 0.998.

Some base fluids might produce a background level that would show up on
the extracted ion profiles, but there should not be any real peaks
(signal to noise ratio of 1:3) from the clean base fluids.

Procedure

Sample Preparation

Mix the authentic field sample (drilling fluid) well. Transfer (weigh) a
30-g aliquot of the sample 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
_Ref239041418 \r \h  \* MERGEFORMAT  6.7.2.3  ), cap with a Teflon lined
crimp cap, and vortex for ca. 10 sec.

The sample is ready for GC/MS analysis.

Gas Chromatography

  REF _Ref238292704 \h  Table 6-1  summarizes the recommended operating
conditions for the GC/MS. Retention times for the n-alkanes obtained
under these conditions are given in   REF _Ref238292711 \h  Table 6-2 .
Other columns, chromatographic conditions, or detectors may be used if
initial precision and accuracy requirements (Section   REF _Ref239038963
\r \h  \* MERGEFORMAT  6.9.2  ) are met. The system shall be calibrated
according to the procedures outlined in Section   REF _Ref239162170 \r
\h  \* MERGEFORMAT  6.10 , and verified every 12 hours according to
Section   REF _Ref239041577 \r \h  \* MERGEFORMAT  6.11.6 .

Samples shall be prepared (extracted) in a batch of no more than 20
samples. The batch shall consist of 20 authentic samples, 1 blank
(Section    REF _Ref239042114 \r \h  \* MERGEFORMAT  6.9.3 ), 1 matrix
spike sample (Section   REF _Ref239042138 \r \h  6.9.4 ), and 1
duplicate field sample (  REF _Ref239042160 \r \h  6.9.5 ), and a
prepared sample of the corresponding clean NAF used in the drilling
process.

An analytical sequence shall be analyzed on the GC/MS where the 3 SPTM
standards (Section   REF _Ref239045057 \r \h  \* MERGEFORMAT  6.7.2.4 )
containing internal standard are analyzed first, followed by analysis of
the four GC/MS crude oil/drilling fluid calibration standards (Section  
REF _Ref239045212 \r \h  \* MERGEFORMAT  6.7.2.5 ), analysis of the
blank, matrix spike sample, the duplicate sample, the clean NAF sample,
followed by the authentic samples.

Samples requiring dilution due to excessive signal shall be diluted
using methylene chloride.

Inject 1.0 µL of the test sample or standard into the GC, using the
conditions in   REF _Ref238292704 \h  Table 6-1 .

Begin data collection and the temperature program at the time of
injection.

Obtain a TIC and EIP fingerprint scans of the sample (  REF
_Ref238292734 \h  Table 6-3 ).

If the area of the C8 to C13 peaks exceeds the calibration range of the
system, dilute a fresh aliquot of the test sample weighing 0.50-g and
re-analyze.

Determine the C8 to C13 TIC area, the TCB internal standard area, and
the areas for the m/z 91 and 105 EIPs. These shall be used in the
calculation of oil concentration in the samples (see Section   REF
_Ref239045581 \r \h  \* MERGEFORMAT  6.12 ).

Table   STYLEREF 1 \s  6 -  SEQ Table \* ARABIC \s 1  3 . Recommended
Ion Mass Numbers

Selected Ion Mass Numbers	Corresponding Aromatic Compounds	Typical
Retention Time (Minutes)

91	Methylbenzene	6.0

	Ethylbenzene	10.3

	1,4-Dimethylbenzene	10.9

	1,3-Dimethylbenzene	10.9

	1,2-Dimethylbenzene	11.9

105	1,3,5-Trimethylbenzene	15.1

	1,2,4-Trimethylbenzene	16.0

	1,2,3-Trimethylbenzene	17.4

156	2,6-Dimethylnaphthalene	28.9

	1,2-Dimethylnaphthalene	29.4

	1,3-Dimethylnaphthalene	29.7



Observe the presence of peaks in the EIPs that would confirm the
presence of any target aromatic compounds. Using the EIP areas and EIP
linear regression calibrations determine approximate crude oil
contamination in the sample for each of the target ions.

Qualitative Identification—See Section 6.17 of this method for
schematic flowchart.

Qualitative identification shall be accomplished by comparison of the
TIC and EIP area data from an authentic sample to the TIC and EIP area
data from the calibration standards (see Section 6.10.4). Crude oil
shall be identified by the presence of C10 to C13 n-alkanes and
corresponding target aromatics.

Using the calibration data, establish the identity of the C8 to C13
peaks in the chromatogram of the sample. Using the calibration data,
establish the identity of any target aromatics present on the extracted
ion scans.

Crude oil is not present in a detectable amount in the sample if there
are no target aromatics seen on the extracted ion scans. The experience
of the analyst shall weigh heavily in the determination of the presence
of peaks at a signal-to-noise ratio of 3 or greater.

If the chromatogram shows n-alkanes from C8 to C13 and target aromatics
to be present, contamination by crude oil or diesel shall be suspected
and quantitative analysis shall be determined. If there are no n-alkanes
present that are not seen on the blank, and no target aromatics are
seen, the sample can be considered to be free of contamination.

Quantitative Identification

Determine the area of the peaks from C8 to C13 as outlined in the
calibration section (Section   REF _Ref239045846 \r \h  6.10.4.1 ). If
the area of the peaks for the sample is greater than that for the clean
NAF (base fluid) use the crude oil/drilling fluid calibration TIC linear
regression curve to determine approximate crude oil contamination.

Using the EIPs outlined in Section   REF _Ref239045888 \r \h  \*
MERGEFORMAT  6.10.4.2 , determine the presence of any target aromatics.
Using the integration techniques outlined in Section   REF _Ref239045888
\r \h  \* MERGEFORMAT  6.10.4.2 , obtain the EIP areas for m/z 91 and
105. Use the crude oil/drilling fluid calibration EIP linear regression
curves to determine approximate crude oil contamination.

Complex Samples

The most common interferences in the determination of crude oil can be
from mineral oil, diesel oil, and proprietary additives in drilling
fluids.

Mineral oil can typically be identified by its lower target aromatic
content, and narrow range of strong peaks.

Diesel oil can typically be identified by low amounts of n-alkanes from
C7 to C9, and the absence of n-alkanes greater than C25.

Crude oils can usually be distinguished by the presence of high
aromatics, increased intensities of C8 to C13 peaks, and/ or the
presence of higher hydrocarbons of C25 and greater (which may be
difficult to see in some synthetic fluids at low contamination levels).

Oil condensates from gas wells are low in molecular weight and will
normally produce strong chromatographic peaks in the C8–C13 range. If
a sample of the gas condensate crude oil from the formation is
available, the oil can be distinguished from other potential sources of
contamination by using it to prepare a calibration standard.

Asphaltene crude oils with API gravity < 20 may not produce
chromatographic peaks strong enough to show contamination at levels of
the calibration. Extracted ion peaks should be easier to see than
increased intensities for the C8 to C13 peaks. If a sample of asphaltene
crude from the formation is available, a calibration standard shall be
prepared.

System and Laboratory Performance

At the beginning of each 8-hour shift during which analyses are
performed, GC crude oil/drilling fluid calibration and system
performance test mixes shall be verified. For these tests, analysis of
the medium-level calibration standard (1-% Reference Oil in IO Lab
drilling fluid, and 1.25 mg/mL SPTM with internal standard) shall be
used to verify all performance criteria. Adjustments and/or
re-calibration (per Section   REF _Ref239046063 \r \h  \* MERGEFORMAT 
6.10 ) shall be performed until all performance criteria are met. Only
after all performance criteria are met may samples and blanks be
analyzed.

Inject 1.0 µL of the medium-level GC/MS crude oil/drilling fluid
calibration standard into the GC instrument according to the procedures
in Section   REF _Ref239046095 \r \h  \* MERGEFORMAT  6.11.2 . Verify
that the linear regression curves for both TIC area and EIP areas are
still valid using this continuing calibration standard.

After this analysis is complete, inject 1.0 µL of the 1.25 mg/mL SPTM
(containing internal standard) into the GC instrument and verify the
proper retention times are met (see   REF _Ref238292711 \h  Table 6-2 ).

Retention times—Retention time of the internal standard. The absolute
retention time of the TCB internal standard shall be within the range
21.0 ±0.5 minutes. Relative retention times of the n-alkanes: The
retention times of the n-alkanes relative to the TCB internal standard
shall be similar to those given in   REF _Ref238292711 \h  Table 6-2 .

Calculations

The concentration of oil in NAFs drilling fluids shall be computed
relative to peak areas between C8 and C13 (using the Total Area
Integration method) or total peak areas from extracted ion profiles
(using the Extracted Ion Profile Method). In either case, there is a
measurable amount of peak area, even in clean drilling fluid samples,
due to spurious peaks and electrometer “noise” that contributes to
the total signal measured using either of the quantification methods. In
this procedure, a correction for this signal is applied, using the blank
or clean sample correction technique described in American Society for
Testing Materials (ASTM) Method D–3328–90, Comparison of Waterborne
Oil by Gas Chromatography. In this method, the “oil equivalents”
measured in a blank sample by total area gas chromatography are
subtracted from that determined for a field sample to arrive at the most
accurate measure of oil residue in the authentic sample.

Total Area Integration Method

Using C8 to C13 TIC area, the TCB area in the clean NAF sample and the
TIC linear regression curve, compute the oil equivalent concentration of
the C8 to C13 retention time range in the clean NAF.

Note: The actual TIC area of the C8 to C13 is equal to the C8 to C13
area minus the area of the TCB.

Using the corresponding information for the authentic sample, compute
the oil equivalent concentration of the C8 to C13 retention time range
in the authentic sample.

Calculate the concentration (% oil) of oil in the sample by subtracting
the oil equivalent concentration (% oil) found in the clean NAF from the
oil equivalent concentration (% oil) found in the authentic sample.

EIP Area Integration Method

Using either m/z 91 or 105 EIP areas, the TCB area in the clean NAF
sample, and the appropriate EIP linear regression curve, compute the oil
equivalent concentration of the in the clean NAF.

Using the corresponding information for the authentic sample, compute
its oil equivalent concentration.

Calculate the concentration (% oil) of oil in the sample by subtracting
the oil equivalent concentration (% oil) found in the clean NAF from the
oil equivalent concentration (% oil) found in the authentic sample.

Method Performance

Specification in this method are adopted from EPA Method 1663,
Differentiation of Diesel and Crude Oil by GC/FID (Section   REF
_Ref239163837 \r \h  6.16 , Reference 4).

Single laboratory method performance using an Internal Olefin (IO)
drilling fluid fortified at 0.5% oil using a 35 API gravity oil was:

Precision and accuracy 94 ±4%

Accuracy interval—86.3% to 102%

Relative percent difference in duplicate analysis—6.2%

Pollution Prevention

The solvent used in this method poses little threat to the environment
when recycled and managed properly.

Waste Management

It is the laboratory's responsibility to comply with all federal, state,
and local regulations governing waste management, particularly the
hazardous waste identification rules and land disposal restriction, and
to protect the air, water, and land by minimizing and controlling all
releases from fume hoods and bench operations. Compliance with all
sewage discharge permits and regulations is also required.

All authentic samples (drilling fluids) failing the RPE (fluorescence)
test (indicated by the presence of fluorescence) shall be retained and
classified as contaminated samples. Treatment and ultimate fate of these
samples is not outlined in this SOP.

For further information on waste management, consult “The Waste
Management Manual for Laboratory Personnel”, and “Less is Better:
Laboratory Chemical Management for Waste Reduction”, both available
from the American Chemical Society's Department of Government Relations
and Science Policy, 1155 16th Street NW, Washington, DC 20036.

References

Carcinogens—“Working With Carcinogens.” Department of Health,
Education, and Welfare, Public Health Service, Centers for Disease
Control (available through National Technical Information Systems, 5285
Port Royal Road, Springfield, VA 22161, document no. PB–277256):
August 1977.

“OSHA Safety and Health Standards, General Industry [29 CFR 1910],
Revised.” Occupational Safety and Health Administration, OSHA 2206.
Washington, DC: January 1976.

“Handbook of Analytical Quality Control in Water and Wastewater
Laboratories.” USEPA, EMSSL-CI, EPA–600/4–79–019. Cincinnati,
OH: March 1979.

“Method 1663, Differentiation of Diesel and Crude Oil by GC/FID,
Methods for the Determination of Diesel, Mineral, and Crude Oils in
Offshore Oil and Gas Industry Discharges, EPA 821–R–92–008, Office
of Water Engineering and Analysis Division, Washington, DC: December
1992.

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).

Schematic Flowchart for Qualitative Identification

                               

	Yes						No

	

                                                                        
                    

                                                                        
  No                   		

                                                                        
                             	

	                                	Yes

	                                

			           	

	

Figure   STYLEREF 1 \s  6 -  SEQ Figure \* ARABIC \s 1  1 .  Schematic
Flowchart for Qualitative Identification

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).

This method 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.

This method 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.

This 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%.

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

Each laboratory that uses this method must demonstrate the ability to
generate acceptable results using the procedure in Section   REF
_Ref239046415 \r \h  \* MERGEFORMAT  7.9.2 .

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.

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).

Interferences

Solvents, reagents, glassware, and other sample-processing hardware may
yield artifacts that affect results. Specific selection of reagents and
purification of solvents may be required.

All materials used in the analysis shall be demonstrated to be free from
interferences under the conditions of analysis by running laboratory
reagent blanks as described in Section   REF _Ref239046495 \r \h  \*
MERGEFORMAT  7.9.5 .

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. Material Safety Data Sheets
(MSDSs) shall be available for all reagents.

Isopropyl alcohol is flammable and should be used in a well-ventilated
area.

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 well-ventilated area to limit the potential exposure
to harmful contaminants. Drilling fluid samples should be handled with
the same precautions used in the drilling fluid handling areas of the
drilling rig.

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 information on laboratory safety
can be found in Section   REF _Ref239164858 \r \h  7.16 , References 1
and 2.

Equipment and Supplies

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 that meets the requirements
of this method is the responsibility of the laboratory.

Sampling equipment.

Sample collection bottles/jars—New, pre-cleaned bottles/jars,
lot-certified to be free of artifacts. Glass preferable, plastic
acceptable, wide mouth approximately 1–L, with Teflon-lined screw cap.

Equipment for glassware cleaning.

Laboratory sink.

Oven—Capable of maintaining a temperature within ±5°C in the range
of 100–250°C.

Equipment for sample extraction.

Vials—Glass, 25 mL and 4 mL, with Teflon-lined screw caps, baked at
200–250°C for 1–h minimum prior to use.

Gas-tight syringes—Glass, various sizes, 0.5 mL to 2.5 mL (if spiking
of drilling fluids with oils is to occur).

Auto pipetters—various sizes, 0.1 mL, 0.5 mL, 1 to 5 mL delivery, and
10 mL delivery, with appropriate size disposable pipette tips,
calibrated to within ±0.5%.

Glass stirring rod.

Vortex mixer.

Disposable syringes—Plastic, 5 mL.

Teflon syringe filter, 25-mm, 0.45 µm pore size—Acrodisc®CR Teflon
(or equivalent).

Reverse Phase Extraction C18Cartridge—Waters Sep-Pak®Plus,
C18Cartridge, 360 mg of sorbent (or equivalent).

SPE vacuum manifold—Supelco Brand, 12 unit (or equivalent). Used as
support for cartridge/syringe assembly only. Vacuum apparatus not
required.

Equipment for fluorescence detection.

Black light—UV Lamp, Model UVG 11, Mineral Light Lamp, Shortwave 254
nm, or Longwave 365 nm, 15 volts, 60 Hz, 0.16 amps (or equivalent).

Black box—cartridge viewing area. A commercially available ultraviolet
viewing cabinet with viewing lamp, or alternatively, a cardboard box or
equivalent, approximately 14 inch × 7.5 inch × 7.5 inch; in size and
painted flat black inside. Lamp positioned in fitted and sealed slot in
center on top of box. Sample cartridges sit in a tray, ca. 6 inches from
lamp. Cardboard flaps cut on top panel and side of front panel for
sample viewing and sample cartridge introduction, respectively.

Viewing platform for cartridges. Simple support (hand made vial
tray—black in color) for cartridges so that they do not move during
the fluorescence testing.

Reagents and Standards

Isopropyl alcohol—99% purity.

NAF—Appropriate NAF as sent from the supplier (has not been circulated
downhole). Use the clean NAF corresponding to the NAF being used in the
current drilling operation.

Standard crude oil—NIST SRM 1582 petroleum crude oil.

Sample Collection, Preservation, and Storage

Collect approximately one liter of representative sample (NAF, which has
been circulated downhole) in a glass bottle or jar. Cover with a Teflon
lined cap. To allow for a potential need to re-analyze and/or re-process
the sample, it is recommended that a second sample aliquot be collected.

Label the sample appropriately.

All samples must be refrigerated at 0–4°C from the time of collection
until extraction (40 CFR Part 136, Table II).

All samples must be analyzed within 28 days of the date and time of
collection (40 CFR Part 136, Table II).

Quality Control

Each laboratory that uses this method is required to operate a formal
quality assurance program (Section   REF _Ref239165058 \r \h  7.16 ,
Reference 3). The minimum requirements of this program consist of an
initial demonstration of laboratory capability, and ongoing analyses of
blanks and spiked duplicates to assess accuracy and precision and to
demonstrate continued performance. Each field sample is analyzed in
duplicate to demonstrate representativeness.

The analyst shall make an initial demonstration of the ability to
generate acceptable accuracy and precision with this method. This
ability is established as described in Section   REF _Ref239046415 \r \h
 \* MERGEFORMAT  7.9.2 .

Preparation and analysis of a set of spiked duplicate samples to
document accuracy and precision. The procedure for the preparation and
analysis of these samples is described in Section   REF _Ref239047094 \r
\h  \* MERGEFORMAT  7.9.4 .

Analyses of laboratory reagent blanks are required to demonstrate
freedom from contamination. The procedure and criteria for preparation
and analysis of a reagent blank are described in Section   REF
_Ref239046495 \r \h  \* MERGEFORMAT  7.9.5 .

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

Accompanying QC for the determination of oil in NAF is required per
analytical batch. An analytical batch is a set of samples extracted at
the same time, to a maximum of 10 samples. Each analytical batch of 10
or fewer samples must be accompanied by a laboratory reagent blank
(Section   REF _Ref239046495 \r \h  \* MERGEFORMAT  7.9.5 ),
corresponding NAF reference blanks (Section   REF _Ref239047193 \r \h 
\* MERGEFORMAT  7.9.6 ), a set of spiked duplicate samples blank
(Section   REF _Ref239047094 \r \h  \* MERGEFORMAT  7.9.4 ), and
duplicate analysis of each field sample. If greater than 10 samples are
to be extracted at one time, the samples must be separated into
analytical batches of 10 or fewer samples.

Initial demonstration of laboratory capability. To demonstrate the
capability to perform the test, the analyst shall analyze two
representative unused drilling fluids (e.g., internal olefin-based
drilling fluid, vegetable ester-based drilling fluid), each prepared
separately containing 0.1%, 1%, and 2% or a representative oil. Each
drilling fluid/concentration combination shall be analyzed 10 times, and
successful demonstration will yield the following average results for
the data set:

0.1% oil—Detected in <20% of samples

1% oil—Detected in >75% of samples

2% oil—Detected in >90% of samples

Sample duplicates.

The laboratory shall prepare and analyze (Section   REF _Ref239047297 \r
\h  \* MERGEFORMAT  7.11.2  and   REF _Ref239047318 \r \h  \*
MERGEFORMAT  7.11.4 ) each authentic sample in duplicate, from a given
sampling site or, if for compliance monitoring, from a given discharge.

The duplicate samples must be compared versus the prepared corresponding
NAF blank.

Prepare and analyze the duplicate samples according to procedures
outlined in Section   REF _Ref239047345 \r \h  \* MERGEFORMAT  7.11 .

The results of the duplicate analyses are acceptable if each of the
results give the same response (fluorescence or no fluorescence). If the
results are different, sample non-homogenicity issues may be a concern.
Prepare the samples again, ensuring a well-mixed sample prior to
extraction. Analyze the samples once again.

If different results are obtained for the duplicate a second time, the
analytical system is judged to be out of control and the problem shall
be identified and corrected, and the samples re-analyzed.

Spiked duplicates—Laboratory prepared spiked duplicates are analyzed
to demonstrate acceptable accuracy and precision.

Preparation and analysis of a set of spiked duplicate samples with each
set of no more than 10 field samples is required to demonstrate method
accuracy and precision and to monitor matrix interferences
(interferences caused by the sample matrix). A field NAF sample expected
to contain less than 0.5% crude oil (and documented to not fluoresce as
part of the sample batch analysis) shall be spiked with 1% (by volume)
of suitable reference crude oil and analyzed as field samples, as
described in Section   REF _Ref239047446 \r \h  \* MERGEFORMAT  7.11 .
If no low-level drilling fluid is available, then the unused NAF can be
used as the drilling fluid sample.

Laboratory reagent blanks—Laboratory reagent blanks are analyzed to
demonstrate freedom from contamination.

A reagent blank is prepared by passing 4 mL of the isopropyl alcohol
through a Teflon syringe filter and collecting the filtrate in a 4-mL
glass vial. A Sep Pak®C18cartridge is then preconditioned with 3 mL of
isopropyl alcohol. A 0.5-mL aliquot of the filtered isopropyl alcohol is
added to the syringe barrel along with 3.0 mL of isopropyl alcohol. The
solvent is passed through the preconditioned Sep Pak® cartridge. An
additional 2-mL of isopropyl alcohol is eluted through the cartridge.
The cartridge is now considered the “reagent blank” cartridge and is
ready for viewing (analysis). Check the reagent blank cartridge under
the black light for fluorescence. If the isopropyl alcohol and filter
are clean, no fluorescence will be observed.

If fluorescence is detected in the reagent blank cartridge, analysis of
the samples is halted until the source of contamination is eliminated
and a prepared reagent blank shows no fluorescence under a black light.
All samples shall be associated with an uncontaminated method blank
before the results may be reported for regulatory compliance purposes.

NAF reference blanks—NAF reference blanks are prepared from the NAFs
sent from the supplier (NAF that has not been circulated downhole) and
used as the reference when viewing the fluorescence of the test samples.

A NAF reference blank is prepared identically to the authentic samples.
Place a 0.1 mL aliquot of the “clean” NAF into a 25-mL glass vial.
Add 10 mL of isopropyl alcohol to the vial. Cap the vial. Vortex the
vial for approximately 10 sec. Allow the solids to settle for
approximately 15 minutes. Using a 5-mL syringe, draw up 4 mL of the
extract and filter it through a PTFE syringe filter, collecting the
filtrate in a 4-mL glass vial. Precondition a Sep Pak® C18 cartridge
with 3 mL of isopropyl alcohol. Add a 0.5-mL aliquot of the filtered
extract to the syringe barrel along with 3.0 mL of isopropyl alcohol.
Pass the extract and solvent through the preconditioned Sep Pak®
cartridge. Pass an additional 2-mL of isopropyl alcohol through the
cartridge. The cartridge is now considered the NAF blank cartridge and
is ready for viewing (analysis). This cartridge is used as the reference
cartridge for determining the absence or presence of fluorescence in all
authentic drilling fluid samples that originate from the same NAF. That
is, the specific NAF reference blank cartridge is put under the black
light along with a prepared cartridge of an authentic sample originating
from the same NAF material. The fluorescence or absence of fluorescence
in the authentic sample cartridge is determined relative to the NAF
reference cartridge.

Positive control solution, equivalent to 1% crude oil contaminated mud
extract, is prepared by dissolving 87 mg of standard crude oil into
10.00 mL of methylene chloride. Then mix 40 µL of this solution into
10.00 mL of IPA. Transfer 0.5 mL of this solution into a preconditioned
C18 cartridge, followed by 2 ml of IPA.

Calibration and Standardization

Calibration and standardization methods are not employed for this
procedure.

Procedure

This method is a screening-level test. Precise and accurate results can
be obtained only by strict adherence to all details.

Preparation of the analytical batch.

Bring the analytical batch of samples to room temperature.

Using a large glass stirring rod, mix the authentic sample thoroughly.

Using a large glass stirring rod, mix the clean NAF (sent from the
supplier) thoroughly.

Extraction.

Using an automatic positive displacement pipetter and a disposable
pipette tip transfer 0.1-mL of the authentic sample into a 25-mL vial.

Using an automatic pipetter and a disposable pipette tip dispense a
10-mL aliquot of solvent grade isopropyl alcohol (IPA) into the 25 mL
vial.

Cap the vial and vortex the vial for ca. 10–15 seconds.

Let the sample extract stand for approximately 5 minutes, allowing the
solids to separate.

Using a 5-mL disposable plastic syringe remove 4 mL of the extract from
the 25-mL vial.

Filter 4 mL of extract through a Teflon syringe filter (25-mm diameter,
0.45 µm pore size), collecting the filtrate in a labeled 4-mL vial.

Dispose of the PFTE syringe filter.

Using a black permanent marker, label a Sep Pak® C18 cartridge with the
sample identification.

Place the labeled Sep Pak® C18 cartridge onto the head of a SPE vacuum
manifold.

Using a 5-mL disposable plastic syringe, draw up exactly 3-mL (air free)
of isopropyl alcohol.

Attach the syringe tip to the top of the C18 cartridge.

Condition the C18 cartridge with the 3-mL of isopropyl alcohol by
depressing the plunger slowly.

Note: Depress the plunger just to the point when no liquid remains in
the syringe barrel. Do not force air through the cartridge. Collect the
eluate in a waste vial.

Remove the syringe temporarily from the top of the cartridge, then
remove the plunger, and finally reattach the syringe barrel to the top
of the C18 cartridge.

Using automatic pipetters and disposable pipette tips, transfer 0.5 mL
of the filtered extract into the syringe barrel, followed by a 3.0-mL
transfer of isopropyl alcohol to the syringe barrel.

Insert the plunger and slowly depress it to pass only the extract and
solvent through the preconditioned C18 cartridge.

Note: Depress the plunger just to the point when no liquid remains in
the syringe barrel. Do not force air through the cartridge. Collect the
eluate in a waste vial.

Remove the syringe temporarily from the top of the cartridge, then
remove the plunger, and finally reattach the syringe barrel to the top
of the C18 cartridge.

Using an automatic pipetter and disposable pipette tip, transfer 2.0 mL
of isopropyl alcohol to the syringe barrel.

Insert the plunger and slowly depress it to pass the solvent through the
C18 cartridge.

Note: Depress the plunger just to the point when no liquid remains in
the syringe barrel. Do not force air through the cartridge. Collect the
eluate in a waste vial.

Remove the syringe and labeled C18 cartridge from the top of the SPE
vacuum manifold.

Prepare a reagent blank according to the procedures outlined in Section 
 REF _Ref239046495 \r \h  \* MERGEFORMAT  7.9.5 .

Prepare the necessary NAF reference blanks for each type of NAF
encountered in the field samples according to the procedures outlined in
Section   REF _Ref239047193 \r \h  \* MERGEFORMAT  7.9.6 .

Prepare the positive control (1% crude oil equivalent) according to
Section   REF _Ref239047796 \r \h  \* MERGEFORMAT  7.9.6.2 .

Reagent blank fluorescence testing.

Place the reagent blank cartridge in a black box, under a black light.

Determine the presence or absence of fluorescence for the reagent blank
cartridge. If fluorescence is detected in the blank, analysis of the
samples is halted until the source of contamination is eliminated and a
prepared reagent blank shows no fluorescence under a black light. All
samples must be associated with an uncontaminated method blank before
the results may be reported for regulatory compliance purposes.

Sample fluorescence testing.

Place the respective NAF reference blank (Section   REF _Ref239047193 \r
\h  \* MERGEFORMAT  7.9.6 ) onto the tray inside the black box.

Place the authentic field sample cartridge (derived from the same NAF as
the NAF reference blank) onto the tray, adjacent and to the right of the
NAF reference blank.

Turn on the black light.

Compare the fluorescence of the sample cartridge with that of the
negative control cartridge (NAF blank, Section   REF _Ref239047881 \r \h
 \* MERGEFORMAT  7.9.6.1 ) and positive control cartridge (1% crude oil
equivalent, Section   REF _Ref239047796 \r \h  \* MERGEFORMAT  7.9.6.2
).

If the fluorescence of the sample cartridge is equal to or brighter than
the positive control cartridge (1% crude oil equivalent, Section   REF
_Ref239047796 \r \h  \* MERGEFORMAT  7.9.6.2  ), the sample is
considered contaminated. Otherwise, the sample is clean.

Data Analysis and Calculations

Specific data analysis techniques and calculations are not performed in
this SOP.

Method Performance

This method was validated through a single laboratory study, conducted
with rigorous statistical experimental design and interpretation
(Section   REF _Ref239165431 \r \h  7.16 , Reference 4).

Pollution Prevention

The solvent used in this method poses little threat to the environment
when recycled and managed properly.

Waste Management

It is the laboratory's responsibility to comply with all Federal, State,
and local regulations governing waste management, particularly the
hazardous waste identification rules and land disposal restriction, and
to protect the air, water, and land by minimizing and controlling all
releases from bench operations. Compliance with all sewage discharge
permits and regulations is also required.

All authentic samples (drilling fluids) failing the fluorescence test
(indicated by the presence of fluorescence) shall be retained and
classified as contaminated samples. Treatment and ultimate fate of these
samples is not outlined in this SOP.

For further information on waste management, consult “The Waste
Management Manual for Laboratory Personnel,” and “Less is Better:
Laboratory Chemical Management for Waste Reduction,” both available
from the American Chemical Society's Department of Government Relations
and Science Policy, 1155 16th Street, NW, Washington, DC 20036.

References

“Carcinogen—Working with Carcinogens,” Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77–206, August 1977.

“OSHA Safety and Health Standards, General Industry,” (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206 (Revised,
January 1976).

“Handbook of Analytical Quality Control in Water and Wastewater
Laboratories,” USEPA, EMSL-Ci, Cincinnati, OH 45268,
EPA–600/4–79–019, March 1979.

Report of the Laboratory Evaluation of Static Sheen Test
Replacements—Reverse Phase Extraction (RPE) Method for Detecting Oil
Contamination in Synthetic Based Mud (SBM). October 1998. Available from
API, 1220 L Street, NW, Washington, DC 20005–4070, 202–682–8000.

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).

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)

Description

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.

In this retort test 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.

Note: Obtaining a representative sample requires special attention to
the details of sample handling (e.g., location, method, frequency). See
Sections   REF _Ref239165594 \r \h  8.5  and   REF _Ref239165602 \r \h 
8.6  for minimum requirements for collecting representative samples.
Additional sampling procedures in a given area may be specified by the
NPDES permit controlling authority.

Equipment

Retort instrument—The recommended retort instrument has a 50-cm3
volume with an external heating jacket.

Retort Specifications:

Retort assembly—retort body, cup and lid.

Material: 303 stainless steel or equivalent.

Volume: Retort cup with lid.

Cup Volume: 50- cm3.

Precision: ±0.25- cm3.

Condenser—capable of cooling the oil and water vapors below their
liquification temperature.

Heating jacket—nominal 350 watts.

Temperature control—capable of limiting temperature of retort to at
least 930°F (500°C) and enough to boil off all NAFs.

Liquid receiver (10- cm3, 20- cm3)—the 10- cm3 and 20- cm3 receivers
are specially designed cylindrical glassware with rounded bottom to
facilitate cleaning and funnel-shaped top to catch falling drops. For
compliance monitoring under the NPDES program, the analyst shall use the
10- cm3 liquid receiver with 0.1 ml graduations to achieve greater
accuracy.

Receiver specifications:

Total volume: 10- cm3, 20- cm3.

Precision (0 to 100%): ±0.05 cm3, ±0.05 cm3.

Outside diameter: 10-mm, 13-mm.

Wall thickness: 1.5 ±0.1mm, 1.2 ±0.1mm.

Frequency of graduation marks (0 to 100%): 0.10- cm3, 0.10- cm3.

Calibration: To contain “TC” @ 20°C.

Scale: cm3, cm3.

Material—Pyrex® or equivalent glass.

Toploading balance—capable of weighing 2,000 g and precision of at
least 0.1 g. Unless motion is a problem, the analyst shall use an
electronic balance. Where motion is a problem, the analyst may use a
triple beam balance.

Fine steel wool (No. 000)—for packing retort body.

Thread sealant lubricant: high temperature lubricant, e.g. Never-Seez®
or equivalent.

Pipe cleaners—to clean condenser and retort stem.

Brush—to clean receivers.

Retort spatula—to clean retort cup.

Corkscrew—to remove spent steel wool.

Procedure

Clean and dry the retort assembly and condenser.

Pack the retort body with steel wool.

Apply lubricant/sealant to threads of retort cup and retort stem.

Weigh and record the total mass of the retort cup, lid, and retort body
with steel wool. This is mass (A), grams.

Collect a representative cuttings sample (see Note in Section   REF
_Ref239048141 \r \h  \* MERGEFORMAT  8.1 ).

Partially fill the retort cup with cuttings and place the lid on the
cup.

Screw the retort cup (with lid) onto the retort body, weigh and record
the total mass. This is mass (B), grams.

Attach the condenser. Place the retort assembly into the heating jacket.

Weigh and record the mass of the clean and dry liquid receiver. This is
mass (C), grams. Place the receiver below condenser outlet.

Turn on the retort. Allow it to run a minimum of 1 hour.

Note: If solids boil over into receiver, the test shall be rerun. Pack
the retort body with a greater amount of steel wool and repeat the test.

Remove the liquid receiver. Allow it to cool. Record the volume of water
recovered. This is (V), cm3.

Note: If an emulsion interface is present between the oil and water
phases, heating the interface may break the emulsion. As a suggestion,
remove the retort assembly from the heating jacket by grasping the
condenser. Carefully heat the receiver along the emulsion band by gently
touching the receiver for short intervals with the hot retort assembly.
Avoid boiling the liquids. After the emulsion interface is broken, allow
the liquid receiver to cool. Read the water volume at the lowest point
of the meniscus.

Weigh and record the mass of the receiver and its liquid contents (oil
plus water). This is mass (D), grams.

Turn off the retort. Remove the retort assembly and condenser from the
heating jacket and allow them to cool. Remove the condenser.

Weigh and record the mass of the cooled retort assembly without the
condenser. This is mass (E), grams.

Clean the retort assembly and condenser.

Calculations

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

Mass of the dry retorted cuttings (MD) equals the mass of the cooled
retort assembly (E) minus the mass of the empty retort assembly (A).

	MD = E − A	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1  2 ]

Mass of the NAF base fluid (MBF) equals the mass of the liquid receiver
with its contents (D) minus the sum of the mass of the dry receiver (C)
and the mass of the water (V).

	MBF = D − (C + V)	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1 
3 ]

Note: Assuming the density of water is 1 g/cm3, the volume of water is
equivalent to the mass of the water.

Mass balance requirement:

The sum of MD, MBF, and V shall be within 5% of the mass of the wet
sample.

 	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1  4 ]

The procedure shall be repeated if this requirement is not met.

Reporting oil from cuttings:

Assume that all oil recovered is NAF base fluid.

The mass percent NAF base fluid retained on the cuttings (%BFi) for the
sampled discharge “i” is equal to 100 times the mass of the NAF base
fluid (MBF) divided by the mass of the wet cuttings sample (Mw).

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

Operators discharging small volume NAF-cuttings discharges which do not
occur during a NAF-cuttings discharge sampling interval (i.e., displaced
interfaces, accumulated solids in sand traps, pit clean-out solids, or
centrifuge discharges while cutting mud weight) shall either: (a)
Measure the mass percent NAF base fluid retained on the cuttings
(%BFSVD) for each small volume NAF-cuttings discharges; or (b) use a
default value of 25% NAF base fluid retained on the cuttings.

The mass percent NAF base fluid retained on the cuttings is determined
for all cuttings wastestreams and includes fines discharges and any
accumulated solids discharged. (See Section   REF _Ref239166859 \r \h 
8.4.3.6  for procedures on measuring or estimating the mass percent NAF
base fluid retained on the cuttings (%BF) for dual gradient drilling
seafloor discharges performed to ensure proper operation of subsea
pumps.)

A mass NAF-cuttings discharge fraction (X, unitless) is calculated for
all NAF-cuttings, fines, or accumulated solids discharges every time a
set of retorts is performed (Section   REF _Ref239166859 \r \h  8.4.3.6 
for procedures on measuring or estimating the mass NAF-cuttings
discharge fraction (X) for dual gradient drilling seafloor discharges
performed to ensure proper operation of subsea pumps). The mass
NAF-cuttings discharge fraction (X) combines the mass of NAF-cuttings,
fines, or accumulated solids discharged from a particular discharge over
a set period of time with the total mass of NAF-cuttings, fines, or
accumulated solids discharged into the ocean during the same period of
time (see Sections   REF _Ref239165630 \r \h  8.5  and   REF
_Ref239165639 \r \h  8.6 ). The mass NAF-cuttings discharge fraction (X)
for each discharge is calculated by direct measurement as:

 	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1  6 ]

Where:

Xi	=	Mass NAF-cuttings discharge fraction for NAF-cuttings, fines, or
accumulated solids discharge “i”, (unitless)

Fi	=	Mass of NAF-cuttings discharged from NAF-cuttings, fines, or
accumulated solids discharge “i” over a specified period of time
(see Sections   REF _Ref239165630 \r \h  8.5  and   REF _Ref239165639 \r
\h  8.6 ), (kg)

G	=	Mass of all NAF-cuttings discharges into the ocean during the same
period of time as used to calculate Fi, (kg)

If an operator has more than one point of NAF-cuttings discharge, the
mass faction (Xi) must be determined by: (a) Direct measurement (see
Equation 8-6); (b) using the following default values of 0.85 and 0.15
for the cuttings dryer (e.g., horizontal centrifuge, vertical
centrifuge, squeeze press, High-G linear shakers) and fines removal unit
(e.g., decanting centrifuges, mud cleaners), respectively, when the
operator is only discharging from the cuttings dryer and the fines
removal unit; or (c) using direct measurement of “Fi” (see Equation
8-6) for fines and accumulated solids, using Equation 8-6A to calculate
“GEST” for use as “G” in Equation 8-6, and calculating the mass
(kg) of NAF-cuttings discharged from the cuttings dryer (Fi) as the
difference between the mass of “GEST” calculated in Equation 8-6A
(kg) and the sum of all fines and accumulated solids mass directly
measured (kg) (see Equation 8-6).

GEST = Estimated mass of all NAF-cuttings discharges into the ocean 

during the same period of time as used to calculate Fi 

	(see Equation 8-6), (kg)	[8-6A]

Where:

GEST	= 	Hole Volume (bbl) × (396.9 kg/bbl) × (1 + Z/100)

Z	= 	The base fluid retained on cuttings limitation or standard (%)
which apply to the NAF being discharge (see 40 CFR§§435.13. and
435.15).

Hole Volume (bbl)	=	[Cross-Section Area of NAF interval (in2)] ×
Average Rate of Penetration (feet/hr) × period of time (min) used to
calculate Fi (see Equation 8-6) × (1 hr/60 min) × (1 bbl/5.61 ft3) ×
(1 ft/12 in)2

Cross-Section Area of NAF interval (in2)	=	(3.14 × [Bit Diameter
(in)]2)/4

Bit Diameter (in)	=	Diameter of drilling bit for the NAF interval
producing drilling cuttings during the same period of time as used to
calculate Fi (see Equation 8-6)

Average Rate of Penetration (feet/hr)	=	Arithmetic average of rate of
penetration into the formation during the same period of time as used to
calculate Fi (see Equation 8-6)



Note: Operators with one NAF-cuttings discharge may set the mass
NAF-cuttings discharge fraction (Xi) equal to 1.0.

Each NAF-cuttings, fines, or accumulated solids discharge has an
associated mass percent NAF base fluid retained on cuttings value (%BF)
and mass NAF-cuttings discharge fraction (X) each time a set of retorts
is performed. A single total mass percent NAF base fluid retained on
cuttings value (%BFT) is calculated every time a set of retorts is
performed. The single total mass percent NAF base fluid retained on
cuttings value (%BFT) is calculated as:

= Σ(Xi) × (%BFi)	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1  7
]

Where:

%BFT,j	= 	Total mass percent NAF base fluid retained on cuttings value
for retort set “j” (unitless as percentage, %)

Xi	= 	Mass NAF-cuttings discharge fraction for NAF-cuttings, fines, or
accumulated solids discharge “i”, (unitless)

%BFi	= 	Mass percent NAF base fluid retained on the cuttings for
NAF-cuttings, fines, or accumulated solids discharge “i” , (unitless
as percentage, %)

Note: ΣXi= 1.

Operators with one NAF-cuttings discharge may set %BFT,j equal to %BFi.

Operators performing dual gradient drilling operations may require
seafloor discharges of large cuttings (>1/4′) to ensure the proper
operation of subsea pumps (e.g., electrical submersible pumps).
Operators performing dual gradient drilling operations which lead to
seafloor discharges of large cuttings for the proper operation of subsea
pumps shall either: (a) Measure the mass percent NAF base fluid retained
on cuttings value (%BF) and mass NAF-cuttings discharge fraction (X) for
seafloor discharges each time a set of retorts is performed; (b) use the
following set of default values, (%BF = 14%; X = 0.15); or (c) use a
combination of (a) and (b) (e.g., use a default value for %BF and
measure X).

Additionally, operators performing dual gradient drilling operations
which lead to seafloor discharges of large cuttings for the proper
operation of subsea pumps shall also perform the following tasks:

Use side scan sonar or shallow seismic to determine the presence of high
density chemosynthetic communities. Chemosynthetic communities are
assemblages of tube worms, clams, mussels, and bacterial mats that occur
at natural hydrocarbon seeps or vents, generally in water depths of 500
meters or deeper. Seafloor discharges of large cuttings for the proper
operation of subsea pumps shall not be permitted within 1,000 feet of a
high density chemosynthetic community.

Seafloor discharges of large cuttings for the proper operation of subsea
pumps shall be visually monitored and documented by a Remotely Operated
Vehicle (ROV) within the tether limit (approximately 300 feet). The
visual monitoring shall be conducted prior to each time the discharge
point is relocated (cuttings discharge hose) and conducted along the
same direction as the discharge hose position. Near-seabed currents
shall be obtained at the time of the visual monitoring.

Seafloor discharges of large cuttings for the proper operation of subsea
pumps shall be directed within a 150 foot radius of the wellbore.

The weighted mass ratio averaged over all NAF well sections (%BFwell) is
the compliance value that is compared with the “maximum weighted mass
ratio averaged over all NAF well sections” BAT discharge limitations
(see the table in 40 CFR §435.13 and footnote 5 of the table in 40 CFR 
§435.43) or the “maximum weighted mass ratio averaged over all NAF
well sections” NSPS discharge limitations (see the table in 40 CFR
§435.15 and footnote 5 of the table in 40 CFR §435.45). The weighted
mass ratio averaged over all NAF well sections (%BFwell) is calculated
as the arithmetic average of all total mass percent NAF base fluid
retained on cuttings values (%BFT) and is given by the following
expression:

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

Where:

%BFwell	= 	Weighted mass ratio averaged over all NAF well sections
(unitless as percentage, %)

%BFT,j	=	Total mass percent NAF base fluid retained on cuttings value
for retort set “j” (unitless as percentage, %)

n 	= 	Total number of retort sets performed over all NAF well sections
(unitless)

Small volume NAF-cuttings discharges which do not occur during a
NAF-cuttings discharge sampling interval (i.e., displaced interfaces,
accumulated solids in sand traps, pit clean-out solids, or centrifuge
discharges while cutting mud weight) shall be mass averaged with the
arithmetic average of all total mass percent NAF base fluid retained on
cuttings values (see Equation 8-8). An additional sampling interval
shall be added to the calculation of the weighted mass ratio averaged
over all NAF well sections (%BFwell). The mass fraction of the small
volume NAF-cuttings discharges (XSVD) will be determined by dividing the
mass of the small volume NAF-cuttings discharges (FSVD) by the total
mass of NAF-cuttings discharges for the well drilling operation (Gwell+
FSVD).

 	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1  9 ]

Where:

XSVD	= 	Mass fraction of the small volume NAF-cuttings discharges
(unitless)

FSVD	= 	Mass of the small volume NAF-cuttings discharges (kg)

Gwell	= 	Mass of total NAF-cuttings from the well (kg)

The mass of small volume NAF-cuttings discharges (FSVD) shall be
determined by multiplying the density of the small volume NAF-cuttings
discharges (ρsvd) times the volume of the small volume NAF-cuttings
discharges (VSVD).

	FSVD = ρsvd × VSVD	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1
 10 ]

Where:

FSVD	= 	Mass of small volume NAF-cuttings discharges (kg)

ρsvd	= 	Density of the small volume NAF-cuttings discharges (kg/bbl)

VSVD	= 	Volume of the small volume NAF-cuttings discharges (bbl)

The density of the small volume NAF-cuttings discharges shall be
measured. The volume of small volume discharges (VSVD) shall be either:
(a) Be measured or (b) use default values of 10 bbl of SBF for each
interface loss and 75 bbl of SBM for pit cleanout per well.

The total mass of NAF-cuttings discharges for the well (Gwell) shall be
either: (a) Measured; or (b) calculated by multiplying 1.0 plus the
arithmetic average of all total mass percent NAF base fluid retained on
cuttings values [see Equation 8-8] times the total hole volume (Vwell)
for all NAF well sections times a default value for the density the
formation of 2.5 g/cm3 (396.9 kg/bbl).

 	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1  11 ]

Where:

Gwell	= 	Total mass of NAF-cuttings discharges for the well (kg)

	= 	see Equation 8-8 (unitless as a percentage)

Vwell	= 	Total hole volume (Vwell) for all NAF well sections (bbl)

The total hole volume of NAF well sections (Vwell) will be calculated
as:

 	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1  12 ]

For wells where small volume discharges associated with cuttings are
made, %BFwell becomes:

 	[  STYLEREF 1 \s  8 -  SEQ Equation \* ARABIC \s 1  13 ]

Note: See Sections   REF _Ref239165630 \r \h  8.5  and   REF
_Ref239165639 \r \h  8.6  to determine the sampling frequency to
determine the total number of retort sets required for all NAF well
sections.

The total number of retort sets (n) is increased by 1 for each sampling
interval (see Section   REF _Ref239059501 \r \h  8.5.2.4 ) when all NAF
cuttings, fines, or accumulated solids for that sampling interval are
retained for no discharge. A zero discharge interval shall be at least
500 feet up to a maximum of three per day. This action has the effect of
setting the total mass percent NAF base fluid retained on cuttings value
(%BFT) at zero for that NAF sampling interval when all NAF cuttings,
fines, or accumulated solids are retained for no discharge.

Operators that elect to use the Best Management Practices (BMPs) for
NAF-cuttings shall use the procedures outlined in Section   REF
_Ref239165834 \r \h  8.6 .

Requirements for Sampling Cuttings Discharge Streams for use with this
Method 

Sampling Locations

Each NAF-cuttings waste stream that discharges into the ocean shall be
sampled and analyzed as detailed earlier in Section   REF _Ref239168941
\r \h  8 . NAF-cuttings discharges to the ocean may include discharges
from primary shakers, secondary shakers, cuttings dryer, fines removal
unit, accumulated solids, and any other cuttings separation device whose
NAF-cuttings waste is discharged to the ocean. NAF-cuttings wastestreams
not directly discharged to the ocean (e.g., NAF-cuttings generated from
shake shakers and sent to a cuttings dryer for additional processing) do
not require sampling and analysis.

The collected samples shall be representative of each NAF-cuttings
discharge. Operators shall conduct sampling to avoid the serious
consequences of error (i.e., bias or inaccuracy). Operators shall
collect NAF-cuttings samples near the point of origin and before the
solids and liquid fractions of the stream have a chance to separate from
one another. For example, operators shall collect shale shaker
NAF-cuttings samples at the point where NAF-cuttings are coming off the
shale shaker and not from a holding container downstream where
separation of larger particles from the liquid can take place.

Operators shall provide a simple schematic diagram of the solids control
system and sample locations to the NPDES permit controlling authority.

Type of Sample and Sampling Frequency

Each NAF-cuttings, fines, or accumulated solids discharge has an
associated mass percent NAF base fluid retained on cuttings value (%BF)
and mass NAF-cuttings discharge fraction (X) for each sampling interval
(see Section   REF _Ref239059501 \r \h  \* MERGEFORMAT  8.5.2.4 ).
Operators shall collect a single discrete NAF-cuttings sample for each
NAF-cuttings waste stream discharged to the ocean during every sampling
interval.

Operators shall use measured depth in feet from the Kelly bushing when
samples are collected.

The NAF-cuttings samples collected for the mass fraction analysis (see
Equation 8-6) shall also be used for the retort analysis (see Equations
8-1 through 8-5).

Operators shall collect and analyze at least one set of NAF-cuttings
samples per day while discharging. Operators engaged in fast drilling
(i.e., greater than 500 linear NAF feet advancement of drill bit per
day) shall collect and analyze one set of NAF-cuttings samples per 500
linear NAF feet of footage drilled. Operators are not required to
collect and analyze more than three sets of NAF-cuttings samples per day
(i.e., three sampling intervals). Operators performing zero discharge of
all NAF-cuttings (i.e., all NAF cuttings, fines, or accumulated solids
retained for no discharge) shall use the following periods to count
sampling intervals: (1) One sampling interval per day when drilling is
less than 500 linear NAF feet advancement of drill bit per day; and (2)
one sampling interval per 500 linear NAF feet of footage drilled with a
maximum of three sampling intervals per day.

The operator shall measure the individual masses (Fi, kg) and sum total
mass (G, kg) (see Equation 8-6) over a representative period of time
(e.g., <10 minutes) during steady-state conditions for each sampling
interval (see Section   REF _Ref239059501 \r \h  \* MERGEFORMAT  8.5.2.4
). The operator shall ensure that all NAF-cuttings are capture for mass
analysis during the same sampling time period (e.g., <10 minutes) at
approximately the same time (i.e., all individual mass samples collected
within one hour of each other).

Operators using Best Management Practices (BMPs) to control NAF-cuttings
discharges shall follow the procedures in Section   REF _Ref239166385 \r
\h  8.6 .

Sample Size and Handling

The volume of each sample depends on the volumetric flow rate (cm3/s) of
the NAF-cuttings stream and the sampling time period (e.g., <10
minutes). Consequently, different solids control equipment units
producing different NAF-cuttings waste streams at different volumetric
flow rates will produce different size samples for the same period of
time. Operators shall use appropriately sized sample containers for each
NAF-cuttings waste stream to ensure no NAF-cuttings are spilled during
sample collection. Operators shall use the same time period (e.g., <10
minutes) to collect NAF-cuttings samples from each NAF-cuttings waste
stream. Each NAF-cuttings sample size shall be at least one gallon.
Operators shall clearly mark each container to identify each
NAF-cuttings sample.

Operators shall not decant, heat, wash, or towel the NAF-cuttings to
remove NAF base fluid before mass and retort analysis.

Operators shall first calculate the mass of each NAF-cuttings sample and
perform the mass ratio analysis (see Equation 8-6). Operators with only
one NAF-cuttings discharge may skip this step (see Section   REF
_Ref239168752 \r \h  8.4.3.4 ).

Operators shall homogenize (e.g., stirring, shaking) each NAF-cuttings
sample prior to placing a sub-sample into the retort cup. The bottom of
the NAF-cuttings sample container shall be examined to be sure that
solids are not sticking to it.

Operators shall then calculate the NAF base fluid retained on cuttings
using the retort procedure (See Equations 8-1 through 8-5). Operators
shall start the retort analyses no more than two hours after collecting
the first individual mass sample for the sampling interval .

Operators shall not discharge any sample before successfully completing
the mass and retort analyses [i.e., mass balance requirements (see
Section   REF _Ref239168784 \r \h  8.4.2 ) are satisfied]. Operators
shall immediately re-run the retort analyses if the mass balance
requirements (see Equation 8-4) are not within a tolerance of 5% (see
Section   REF _Ref239168784 \r \h  8.4.2 , Equation 8-4).

Calculations

Operators shall calculate a set of mass percent NAF base fluid retained
on cuttings values (%BF) and mass NAF-cuttings discharge fractions (X)
for each NAF-cuttings waste stream (see Section   REF _Ref239060447 \r
\h  \* MERGEFORMAT  8.5.1.1 ) for each sampling interval (see Section  
REF _Ref239059501 \r \h  \* MERGEFORMAT  8.5.2.4 ) using the procedures
outlined earlier in Section   REF _Ref239168918 \r \h  8 .

Operators shall tabulate the following data for each individual
NAF-cuttings sample: (1) Date and time of NAF-cuttings sample
collection; (2) time period of NAF-cuttings sample collection (see
Section   REF _Ref239060572 \r \h  \* MERGEFORMAT  8.5.3.1 ); (3) mass
and volume of each NAF-cuttings sample; (4) measured depth (feet) at
NAF-cuttings sample collection (see Section   REF _Ref239060636 \r \h 
\* MERGEFORMAT  8.5.2.2 ); (5) respective linear feet of hole drilled
represented by the NAF-cuttings sample (feet); and (6) the drill bit
diameter (inches) used to generate the NAF-cuttings sample cuttings.

Operators shall calculate a single total mass percent NAF base fluid
retained on cuttings value (%BFT) for each sampling interval (see
Section   REF _Ref239059501 \r \h  \* MERGEFORMAT  8.5.2.4 ) using the
procedures outlined in Section   REF _Ref239169625 \r \h  8.4 .

Operators shall tabulate the following data for each total mass percent
NAF base fluid retained on cuttings value (%BFT) for each NAF-cuttings
sampling interval: (1) Date and starting and stopping times of
NAF-cuttings sample collection and retort analyses; (2) measured depth
of well (feet) at start of NAF-cuttings sample collection (see Section  
REF _Ref239060636 \r \h  \* MERGEFORMAT  8.5.2.2 ); (3) respective
linear feet of hole drilled represented by the NAF-cuttings sample
(feet); (4) the drill bit diameter (inches) used to generate the
NAF-cuttings sample cuttings; and (5) annotation when zero discharge of
NAF-cuttings is performed.

Operators shall calculate the weighted mass ratio averaged over all NAF
well sections (%BFwell) using the procedures outlined in Section   REF
_Ref239169625 \r \h  8.4 .

Operators shall tabulate the following data for each weighted mass ratio
averaged over all NAF well sections (%BFwell) for each NAF well: (1)
Starting and stopping dates of NAF well sections; (2) measured depth
(feet) of all NAF well sections; (3) total number of sampling intervals
(see Sections   REF _Ref239059501 \r \h  \* MERGEFORMAT  8.5.2.4  and  
REF _Ref239060748 \r \h  \* MERGEFORMAT  8.5.2.6 ); (4) number of
sampling intervals tabulated during any zero discharge operations; (5)
total volume of zero discharged NAF-cuttings over entire NAF well
sections; and (6) identification of whether BMPs were employed (see
Section   REF _Ref239168973 \r \h  8.6 ).

Best Management Practices (BMPs) for use with this Method 

Overview of BMPs

Best Management Practices (BMPs) are inherently pollution prevention
practices. BMPs may include the universe of pollution prevention
encompassing production modifications, operational changes, material
substitution, materials and water conservation, and other such measures.
BMPs include methods to prevent toxic and hazardous pollutants from
reaching receiving waters. Because BMPs are most effective when
organized into a comprehensive facility BMP Plan, operators shall
develop a BMP in accordance with the requirements in this addendum.

The BMP requirements contained in this section were compiled from
several Regional permits, an EPA guidance document (i.e., Guidance
Document for Developing Best Management Practices (BMP)” (EPA
833–B–93–004, U.S. EPA, 1993)), and draft industry BMPs. These
common elements represent the appropriate mix of broad directions needed
to complete a BMP Plan along with specific tasks common to all drilling
operations.

Operators are not required to use BMPs if all NAF-cuttings discharges
are monitored in accordance with Sections   REF _Ref239169012 \r \h  8.1
 through   REF _Ref239169025 \r \h  8.4 .

BMP Plan Purpose and Objectives

Operators shall design the BMP Plan to prevent or minimize the
generation and the potential for the discharge of NAF from the facility
to the waters of the United States through normal operations and
ancillary activities. The operator shall establish specific objectives
for the control of NAF by conducting the following evaluations.

The operator shall identify and document each NAF well that uses BMPs
before starting drilling operations and the anticipated total feet to be
drilled with NAF for that particular well.

Each facility component or system controlled through use of BMPs shall
be examined for its NAF-waste minimization opportunities and its
potential for causing a discharge of NAF to waters of the United States
due to equipment failure, improper operation, natural phenomena (e.g.,
rain, snowfall).

For each NAF wastestream controlled through BMPs where experience
indicates a reasonable potential for equipment failure (e.g., a tank
overflow or leakage), natural condition (e.g., precipitation), or other
circumstances to result in NAF reaching surface waters, the BMP Plan
shall include a prediction of the total quantity of NAF which could be
discharged from the facility as a result of each condition or
circumstance.

BMP Plan Requirements

The BMP Plan may reflect requirements within the pollution prevention
requirements required by the Minerals Management Service (see 30 CFR
250.300) or other Federal or State requirements and incorporate any part
of such plans into the BMP Plan by reference.

The operator shall certify that its BMP Plan is complete, on-site, and
available upon request to EPA or the NPDES Permit controlling authority.
This certification shall identify the NPDES permit number and be signed
by an authorized representative of the operator. This certification
shall be kept with the BMP Plan. For new or modified NPDES permits, the
certification shall be made no later than the effective date of the new
or modified permit. For existing NPDES permits, the certification shall
be made within one year of permit issuance.

The BMP Plan shall:

Be documented in narrative form, and shall include any necessary plot
plans, drawings or maps, and shall be developed in accordance with good
engineering practices. At a minimum, the BMP Plan shall contain the
planning, development and implementation, and evaluation/reevaluation
components. Examples of these components are contained in “Guidance
Document for Developing Best Management Practices (BMP)” (EPA
833–B–93–004, U.S. EPA, 1993).

Include the following provisions concerning BMP Plan review.

Be reviewed by permittee's drilling engineer and offshore installation
manager (OIM) to ensure compliance with the BMP Plan purpose and
objectives set forth in Section   REF _Ref239061899 \r \h  \*
MERGEFORMAT  8.6.2 .

Include a statement that the review has been completed and that the BMP
Plan fulfills the BMP Plan purpose and objectives set forth in Section  
REF _Ref239061899 \r \h  \* MERGEFORMAT  8.6.2 . This statement shall
have dated signatures from the permittee's drilling engineer and
offshore installation manager and any other individuals responsible for
development and implementation of the BMP Plan.

Address each component or system capable of generating or causing a
release of significant amounts of NAF and identify specific preventative
or remedial measures to be implemented.

BMP Plan Documentation

The operator shall maintain a copy of the BMP Plan and related
documentation (e.g., training certifications, summary of the monitoring
results, records of NAF-equipment spills, repairs, and maintenance) at
the facility and shall make the BMP Plan and related documentation
available to EPA or the NPDES Permit controlling authority upon request.

BMP Plan Modification

For those NAF wastestreams controlled through BMPs, the operator shall
amend the BMP Plan whenever there is a change in the facility or in the
operation of the facility which materially increases the generation of
those NAF-wastes or their release or potential release to the receiving
waters.

At a minimum the BMP Plan shall be reviewed once every five years and
amended within three months if warranted. Any such changes to the BMP
Plan shall be consistent with the objectives and specific requirements
listed in this addendum. All changes in the BMP Plan shall be reviewed
by the permittee's drilling engineer and offshore installation manager.

At any time, if the BMP Plan proves to be ineffective in achieving the
general objective of preventing and minimizing the generation of
NAF-wastes and their release and potential release to the receiving
waters and/or the specific requirements in this addendum, the permit
and/or the BMP Plan shall be subject to modification to incorporate
revised BMP requirements.

Specific Pollution Prevention Requirements for NAF Discharges Associated
with Cuttings

The following specific pollution prevention activities are required in a
BMP Plan when operators elect to control NAF discharges associated with
cuttings by a set of BMPs.

Establishing programs for identifying, documenting, and repairing
malfunctioning NAF equipment, tracking NAF equipment repairs, and
training personnel to report and evaluate malfunctioning NAF equipment.

Establishing operating and maintenance procedures for each component in
the solids control system in a manner consistent with the manufacturer's
design criteria.

Using the most applicable spacers, flushes, pills, and displacement
techniques in order to minimize contamination of drilling fluids when
changing from water-based drilling fluids to NAF and vice versa.

A daily retort analysis shall be performed (in accordance with Sections 
 REF _Ref239169371 \r \h  8.1  through   REF _Ref239169382 \r \h  8.4 )
during the first 0.33 X feet drilled with NAF where X is the anticipated
total feet to be drilled with NAF for that particular well. The retort
analyses shall be documented in the well retort log. The operators shall
use the calculation procedures detailed in Section   REF _Ref239169382
\r \h  8.4  (see Equations 8-1 through 8-8) to determine the arithmetic
average (%BFwell) of the retort analyses taken during the first 0.33 X
feet drilled with NAF.

When the arithmetic average (%BFwell) of the retort analyses taken
during the first 0.33 X feet drilled with NAF is less than or equal to
the base fluid retained on cuttings limitation or standard (see 40 CFR
§§435.13 and 435.15), retort monitoring of cuttings may cease for that
particular well. The same BMPs and drilling fluid used during the first
0.33 X feet shall be used for all remaining NAF sections for that
particular well.

When the arithmetic average (%BFwell) of the retort analyses taken
during the first 0.33 X feet drilled with NAF is greater the base fluid
retained on cuttings limitation or standard (see 40 CFR §§435.13 and
435.15), retort monitoring shall continue for the following (second)
0.33 X feet drilled with NAF where X is the anticipated total feet to be
drilled with NAF for that particular well. The retort analyses for the
first and second 0.33 X feet shall be documented in the well retort log.

When the arithmetic average (%BFwell) of the retort analyses taken
during the first 0.66 X feet (i.e., retort analyses taken from first and
second 0.33 X feet) drilled with NAF is less than or equal to the base
fluid retained on cuttings limitation or standard (see 40 CFR §§435.13
and 435.15), retort monitoring of cuttings may cease for that particular
well. The same BMPs and drilling fluid used during the first 0.66 X feet
shall be used for all remaining NAF sections for that particular well.

When the arithmetic average (%BFwell) of the retort analyses taken
during the first 0.66 X feet (i.e., retort analyses taken from first and
second 0.33 X feet) drilled with NAF is greater than the base fluid
retained on cuttings limitation or standard (see 40 CFR §§435.13 and
435.15), retort monitoring shall continue for all remaining NAF sections
for that particular well. The retort analyses for all NAF sections shall
be documented in the well retort log.

When the arithmetic average (%BFwell) of the retort analyses taken over
all NAF sections for the entire well is greater that the base fluid
retained on cuttings limitation or standard (see 40 CFR §§435.13 and
435.15), the operator is in violation of the base fluid retained on
cuttings limitation or standard and shall submit notification of these
monitoring values in accordance with NPDES permit requirements.
Additionally, the operator shall, as part of the BMP Plan, initiate a
reevaluation and modification to the BMP Plan in conjunction with
equipment vendors and/or industry specialists.

The operator shall include retort monitoring data and dates of
retort-monitored and non-retort-monitored NAF-cuttings discharges
managed by BMPs in their NPDES permit reports.

Establishing mud pit and equipment cleaning methods in such a way as to
minimize the potential for building-up drill cuttings (including
accumulated solids) in the active mud system and solids control
equipment system. These cleaning methods shall include but are not
limited to the following procedures.

Ensuring proper operation and efficiency of mud pit agitation equipment.

Using mud gun lines during mixing operations to provide agitation in
dead spaces.

Pumping drilling fluids off of drill cuttings (including accumulated
solids) for use, recycle, or disposal before using wash water to
dislodge solids.

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.

This method is for use in the Environmental Protection Agency’s
(EPA’s) survey and monitoring programs under the Federal Water
Pollution Control Act.

For oil in drilling muds, this method is designed to be used in
conjunction with the extraction procedure in EPA Method 1662.

The level of PAH in   REF _Ref238902725 \h  Table 9-1  typifies the
minimum level that can be detected in oil with this method.

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.

This method is restricted to use by or under the supervision of analysts
experienced in the use of HPLC systems and in the interpretation of
liquid chromatograms. Each analyst must demonstrate the ability to
generate acceptable results with this method using the procedure
described in Section   REF _Ref238899892 \r \h  9.8.2 .

Summary of Method

d 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.

Quality is assured through reproducible calibration and testing of the
extraction and HPLC systems.

Interferences

Solvents, reagents, glassware, and other sample processing hardware may
lead to discrete 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 initially and with each sample
batch (samples started through the extraction process at the same time,
to a maximum of ten). Specific selection of reagents and purification of
solvents by distillation in all-glass systems may be required.

Glassware and, where possible, reagents are cleaned by solvent rinse
and/or baking at 450°C for a minimum of 1 hour.

When used in conjunction with Method 1662, blanks extracted in that
method are treated as an integral part of this method.

Interferences co-extracted from samples may vary from source to source,
depending on the diversity of the site being sampled.

Safety

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

The laboratory is responsible for maintaining 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) should also be made available to all personnel involved
in the chemical analysis. Additional information on laboratory safety
can be found in References 1 through 3.

Methylene chloride has been classified as a known health hazard. All
steps in this method which involve exposure to this compound shall be
per-formed in an OSHA-approved fume hood.

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, Sante 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 i.d. (Fisher
Scientific 13-678-6A, or equivalent).

Glass pipettes: 1.0- and 10-mL, accurate to 1 % or better.

Volumetric flasks: Glass, 10- and 100-mL.

Sample vials: Amber glass, 2- to 5-mL with PTFE-lined screw-cap, to fit
HPLC autosampler.

High-performance liquid chromatograph (HPLC): An analytical system
complete with pumps, sample injector, column oven, and ultra-violet (UV)
detector.

Pumping system: Capable of isocratic operation and producing a linear
gradient from 50% water/50% acetonitrile to 100% acetonitrile in 10
minutes (Waters 600E, or equivalent).

Sample injector: Capable of automated injection of up to 30 samples
(Waters 700, or equivalent).

Column oven: Capable of operation at room ambient to 50°C (Waters TCM,
or equivalent).

Column: Two C18 columns, 150 mm long by 4.6 mm i.d., 300 angstroms
(Vydac 201 TP5415, or equivalent) connected in series, preceded by one
C18 guard column, 30 mm long by 4.6 mm i.d., 300 angstroms (Vydac 201
GCC54T, or equivalent), operated at the conditions shown in   REF
_Ref238902725 \h  Table 9-1 .

Detector: UV operated at 254 nm (Waters 490E, or equivalent).

Data system.

Data acquisition: The data system shall collect and record LC peak areas
and retention times on magnetic media.

Calibration: The data system shall be used to calculate and maintain
lists of calibration factors (response divided by concentration) and
multi-point calibration curves. Computations of relative standard
deviation (coefficient of variation) are used to test calibration
linearity.

Data processing: The data system shall be used to search, locate,
identify, and quantify the compounds of interest in each analysis.
Displays of chromatograms are required to verify results.

Statistics on initial (Section   REF _Ref238899892 \r \h  9.8.2 ) and
ongoing (Section   REF _Ref238900267 \r \h  9.12.6 ) performance shall
be computed and maintained.

Reagents

Solvents.

Sample preparation: Methylene chloride, distilled in glass (Burdick and
Jackson, or equivalent).

HPLC: Methanol, acetonitrile, and water, HPLC quality.

Standards: Purchased as solutions or mixtures with certification to
their purity, concentration, and authenticity, or prepared from
materials of known purity and composition. If compound purity is 96% or
greater, the weight may be used without correction to compute the
concentration of the standard. If PAH in oil from drilling mud is to be
tested, the diesel oil standard used in this method should be from the
oil used on the drilling rig from which the mud sample is taken. If this
oil is not available, No. 2 diesel oil from a local source may be
substituted.

Stock solutions: Prepare in methylene chloride or methanol and dilute In
acetonitrile for injection into the HPLC. Observe the safety precautions
in Section   REF _Ref238900320 \r \h  9.4 .

Diesel oil solutions

Stock solution in methylene chloride (62.5 mg/mL): If QC extracts from
Method 1662 are to be tested, use the oil that was spiked in that
method. Weigh 6.25 g of diesel oil into a 100-mL ground-glass-stoppered
volumetric flask and fill to the mark with methylene chloride.

Diesel oil calibration solution (1.25 mg/mL): After the oil in the stock
solution (see bullet above) is completely dissolved, remove 1.00 mL and
place in a 50-mL volumetric flask. Dilute to the mark with acetonitrile.
Mix thoroughly and transfer to a clean 150mL bottle with PTFE-lined cap.

Polynuclear aromatic hydrocarbons-naphthalene, phenanthrene, and
indeno[1,2,3-cd]pyrene: Dissolve an appropriate amount of reference
material in a suitable solvent. For example, weigh 10.0 mg of
naphthalene in a 10-mL volumetric flask and fill to the mark with
methanol. After the naphthalene is completely dissolved, transfer the
solution to a 15-mL vial with PTFE-lined cap.

Stock solutions should be checked for signs of degradation prior to the
preparation of calibration or performance test standards.

PAH calibration standards (CAL): Dilute and mix the stock solutions
(Section   REF _Ref238900613 \r \h  9.6.2.1b ) in acetonitrile to
produce the calibration standards shown in   REF _Ref238900719 \h  Table
9-2 . The three solutions permit the response of phenanthrene to be
measured as a function of concentration, and naphthalene and
indeno[1,2,3-cd]pyrene permit the retention time window for PAH to be
defined. The medium-level solution is used for calibration verification
(Section   REF _Ref238900803 \r \h  9.12.2 ).

Precision and recovery standard: The diesel oil calibration solution
(Section   REF _Ref238900918 \r \h  \* MERGEFORMAT  9.6.2.1a ), second
bullet) is used for initial precision and recovery (IPR; Section   REF
_Ref238899892 \r \h  \* MERGEFORMAT  9.8.2 ) and ongoing precision and
recovery (OPR, Section   REF _Ref238900267 \r \h  \* MERGEFORMAT  9.12.6
).

Stability of solutions.

When not being used, standards are stored in the dark at -20 to -10°C
in screw-capped vials with PTFE-lined lids. A mark is placed on the vial
at the level of the solution so that solvent loss by evaporation can be
detected. The vial is brought to room temperature prior to use. Any
precipitate is redissolved and solvent is added if solvent loss has
occurred.

Standard solutions used for quantitative purposes (Sections   REF
_Ref238901363 \r \h  \* MERGEFORMAT  9.6.2.1  through   REF
_Ref238901381 \r \h  \* MERGEFORMAT  9.6.2.3 ) shall be analyzed within
48 hours of preparation and on a monthly basis thereafter for signs of
degradation. Standards will remain
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Inject 20 μL- of the medium level calibration standard (  REF
_Ref238900719 \h  Table 9-2 ).

Locate the three peaks in this standard.

Adjust the initial solvent mixture, the isocratic hold, the gradient,
and the final isocratic hold until the retention times are within + 1
minute of the retention times given in   REF _Ref238900719 \h  Table 9-2
.

Minimum level: Analyze 20 μL of the low-level calibration standard ( 
REF _Ref238900719 \h  Table 9-2 ) and verify that the HPLC instrument
meets the minimum level for phenanthrene in   REF _Ref238902725 \h 
Table 9-1 .

External standard calibration.

Analyze 20 μL of each calibration standard (  REF _Ref238900719 \h 
Table 9-2 ) beginning with the lowest concentration and proceeding to
the highest using to the procedure in Section   REF _Ref238901537 \r \h 
9.11 .

Record the areas for the phenanthrene peak and the height of the
phenanthrene peak in the high-level standard.

Compute the ratio of response to amount injected (calibration factor) at
each concentration by dividing the area of the peak by the concentration
of the standard injected. Calculate the mean of the three values to
produce an average calibration factor.

Linearity: If the calibration factor is constant over the three point
calibration range (< 15% relative standard deviation), linearity through
the origin can be assumed; if not, the system shall be recalibrated.

The average calibration factor is verified on each working 8-hour shift
by the measurement of the medium-level calibration standard (Section  
REF _Ref238901565 \r \h  \* MERGEFORMAT  9.12.5 ).

Single-point calibration for diesel oil: Inject the precision and
recovery standard (Section   REF _Ref238901381 \r \h  \* MERGEFORMAT 
9.6.2.3 ) to produce a single calibration point for diesel oil.

Integrate the area from the retention time of naphthalene (including the
leading edge of the naphthalene peak) through the end of the
indeno[1,2,3-cd]pyrene peak or until the detector signal returns to a
stable baseline, whichever comes later, as shown in   REF _Ref238901607
\h  Figure 9-1 .

Determine the calibration factor for diesel oil by dividing the
integrated area (  REF _Ref238901636 \r \h  9.7.6.1 ) by the diesel oil
concentration (Section   REF _Ref238900918 \r \h  \* MERGEFORMAT 
9.6.2.1a ), second bullet).

Quality Assurance/Quality Control

Each laboratory that uses this method is required to operate a formal
quality assurance program (Reference 4). The minimum requirements of
this program consist of an initial demonstration of laboratory
capability, an 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
is compared to established performance criteria to determine if the
results of analyses meet the performance characteristics of the method.
If the determination of PAH is to be made on extracts from Method 1662,
the quality control samples for initial precision and recovery (IPR),
spiked samples, duplicate samples, and ongoing precision and recovery
(OPR) samples from Method 1662 shall be substituted for those in the QC
tests below, and the specifications in   REF _Ref238902725 \h  Table 9-1
 for extracts from Method 1662 shall be met.

The analyst shall make an initial demonstration of the ability to
generate acceptable accuracy and precision with this method. This
ability is established as described in Section   REF _Ref238899892 \r \h
 9.8.2 .

The analyst is permitted to modify this method to improve separations or
lower the costs of measurements, provided all performance requirements
are met. Each time a modification is made to the method, the analyst is
required to achieve the minimum level (Section   REF _Ref238902775 \r \h
 9.7.3 ) and to repeat the procedure in Section   REF _Ref238899892 \r
\h  9.8.2  to demonstrate method performance.

Analyses of spiked samples are required to demonstrate method accuracy
when extracts from Method 1662 are analyzed. The procedure and QC
criteria for spiking are described in Section   REF _Ref238902806 \r \h 
9.8.3 .

Analyses of duplicate samples are required to demonstrate method
precision when extracts from Method 1662 are analyzed. The procedure and
QC criteria for duplicates are described in   REF _Ref238902823 \r \h 
9.8.4 .

Analyses of blanks are required to demonstrate freedom from
contamination. The procedures and criteria for analysis of a blank are
described in Section   REF _Ref238902861 \r \h  9.8.5 .

The laboratory shall, on an ongoing basis, demonstrate through
calibration verification and analysis of the precision and recovery
standard that the analysis system is in control. These procedures are
described in Sections   REF _Ref238901565 \r \h  \* MERGEFORMAT  9.12.5 
and   REF _Ref238900267 \r \h  \* MERGEFORMAT  9.12.6 .

The laboratory shall maintain records to define the quality of data that
is generated. Development of accuracy statements is described in
Sections   REF _Ref238905855 \r \h  9.8.3.2  and   REF _Ref238905867 \r
\h  9.12.6.4 .

Initial precision and recovery (IPR): The initial precision and recovery
test is performed using the precision and recovery standard. If extracts
from Method 1662 are to be analyzed, the extracts from the initial
precision and recovery tests in that method shall be used; otherwise,
the laboratory shall generate acceptable precision and recovery by
performing the following operations.

Using diesel oil, prepare four separate aliquots of the precision and
recovery standard (Section   REF _Ref238901381 \r \h  9.6.2.3 ). If
extracts from Method 1662 are analyzed, the extracts from the initial
precision and recovery test in that method shall be used. Analyze these
aliquots using the procedure in Section   REF _Ref238905895 \r \h  9.11
.

Using results of the set of four analyses, compute the average recovery
(X) of PAH in mg/mL and the standard deviation of the recovery (s) in
mg/mL for each aliquot by the external standard method (Sections   REF
_Ref238905919 \r \h  \* MERGEFORMAT  9.7.4  and   REF _Ref238905932 \r
\h  \* MERGEFORMAT  9.14.4 ).

Compare s and X with the corresponding limits for initial precision and
recovery in   REF _Ref238902725 \h  Table 9-1 . If s and X meet the
acceptance criteria, system performance is acceptable and analysis of
oil 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.

Method accuracy: If extracts from Method 1662 are to be analyzed, the
extract from the accuracy test in that method shall be used; otherwise,
an accuracy test is unnecessary. The procedure for determining method
accuracy is given in Section 8.3 of Method 1662, and the specification
for accuracy is given in   REF _Ref238902725 \h  Table 9-1  of this
method.

Compare the percent recovery of PAH with the corresponding QC acceptance
criteria in   REF _Ref238902725 \h  Table 9-1 . If the results of the
spike fail the acceptance criteria, and the recovery of the QC standard
in the ongoing precision and recovery test (Section   REF _Ref238906537
\r \h  9.12.6.3 ) is within the acceptance criteria in   REF
_Ref238902725 \h  Table 9-1 , an interference may be present. In this
case, the result may not be reported for regulatory compliance purposes.
If, however, the results of both the spike and the ongoing precision and
recovery test fail the acceptance criteria, the analytical system is
judged to be out of control and the problem shall be identified and
corrected, and the sample batch reanalyzed.

As part of the QA program for the laboratory, method accuracy for
samples shall be assessed and records shall be maintained. After the
analysis of five spiked samples in which the recovery passes the test in
Section   REF _Ref238902806 \r \h  9.8.3 , compute the average percent
recovery (P) and the standard deviation of the percent recovery (P).
Express the accuracy assessment as a percent recovery interval from P
– 2Sp to P + 2 Sp. For example, if P = 90% and Sp = 10% for five
analyses of PAH in diesel oil, the accuracy interval is expressed as 70
to 110%. Update the accuracy assessment on a regular basis (e.g., after
each five to ten new accuracy measurements).

Duplicates: If extracts from Method 1662 are to be analyzed, the
extracts from the duplicates test in that method shall be used. The
procedure for preparing duplicates is given in Section 8.4 of Method
1662, and the specification for RPD is given in   REF _Ref238902725 \h 
\* MERGEFORMAT  Table 9-1  of this method. If extracts from Method 1662
are not to be analyzed, duplicates of the precision and recovery
standard (Section   REF _Ref238901381 \r \h  9.6.2.3 ) are analyzed, and
the specification for RPD is given for PAH in diesel oil in   REF
_Ref238902725 \h  Table 9-1  of this method.

Analyze each of the duplicates per the procedure in Section   REF
_Ref238906598 \r \h  9.11  and compute the results per Section   REF
_Ref238906610 \r \h  9.14 .

Calculate the relative percent difference (RPD) between the two results
per the following equation:

 	[  STYLEREF 1 \s  9 -  SEQ Equation \* ARABIC \s 1  1 ]

Where:

D1	=	Concentration of diesel oil in the sample

D2	=	Concentration of diesel oil in the second (duplicate) sample

The relative percent difference for duplicates shall meet the acceptance
criteria in   REF _Ref238902725 \h  Table 9-1 . If the criteria are not
met, the analytical system is be judged to be out of control, and the
problem must be immediately identified and corrected and the sample set
re-extracted and reanalyzed.

Blanks: If extracts from Method 1662 are to be analyzed, the extracts
from blanks in that method shall be analyzed in addition to the blanks
in this method.

Rinse the glassware used in preparation of the extracts in this method
with acetonitrile and analyze a 20-μL aliquot of the rinsate using the
procedure in Section   REF _Ref238906628 \r \h  9.11  and compute the
results per Section   REF _Ref238906639 \r \h  9.14 .

If PAH is detected in a blank at greater than the method detection limit
(MDL) in   REF _Ref238902725 \h  Table 9-1 , analysis of samples is
halted until the source of contamination is eliminated and a blank shows
no evidence of contamination.

The specifications contained in this method can be met if the apparatus
used is calibrated properly, then maintained in a calibrated state. The
standards used for initial precision and recovery (IPR, Section   REF
_Ref238899892 \r \h  9.8.2 ) and ongoing precision and recovery (OPR,
Section   REF _Ref238900267 \r \h  9.12.6 ) should be identical, so that
the most precise results will be obtained. The HPLC instrument will
provide the most reproducible results if dedicated to the settings and
conditions required for the analyses given in this method.

Depending on specific program requirements, field replicates and field
spikes of diesel oil into samples may be required when Method 1662 and
this method are used to assess the precision and accuracy of the
sampling and sample transportation techniques.

Sample Collection, Preservation, and Handling

Oil samples are collected in 20- to 40-mL vials with PTFE- or
aluminum-foil-lined caps and stored in the dark at -20 to -10°C.

If extracts from Method 1662 are to be analyzed, the laboratory should
be aware that sample and extract holding times for this method have not
yet been established. However, based on tests of wastewater for the
analytes determined in this method, samples shall be extracted within 7
days of collection and extracts shall be analyzed within 40 days of
extraction.

As a precaution against analyte and solvent loss or degradation, sample
extracts are stored in glass bottles with PTFE-lined caps, in the dark,
at -20 to -10°C.

Dilution of Oil and Extracts

000 μg/mL. Mineral oils and other oils containing a lesser PAH content
will require less dilution.

Extracts from Method 1662: If extracts of samples from Method 1662 are
to be analyzed, these extracts (from Section 10.4.2 of that method) are
analyzed undiluted unless diesel oil is known or suspected to be
present. Extracts of QC samples (IPR, OPR, matrix spikes, and
duplicates) from Method 1662 are diluted by a factor of 10 to bring them
within the range of the HPLC.

Dilution of neat oil expected to be diesel oil.

Weigh 100 mg into a 10-mL volumetric flask and dilute to the mark with
methylene chloride to produce a concentration of 10 mg/mL. Stopper and
mix thoroughly.

Using a calibrated 1.0-mL volumetric pipette, withdraw 1.0 mL of the
solution and place in a 10-mL volumetric flask. Then withdraw an
additional 0.25 mL of the solution and place in the 10-mL volumetric
flask (for a total of 1.25 mL). Fill to the mark with acetonitrile to
produce a concentration of 1.25 mg/mL (1250 μg/mL). This solution will
be near, but not above, the limit of the calibration range and will
match the concentration of the QC samples from Method 1662 (assuming
100% recovery).

High-Performance Liquid Chromatography

  REF _Ref238900719 \h  Table 9-2  summarizes the recommended operating
conditions for the HPLC. Included in this table and in   REF
_Ref238902725 \h  Table 9-1  are retention times and the minimum level
that can be achieved under these conditions. An example of the
separation achieved for diesel oil by the multiple HPLC column system is
shown in   REF _Ref238901607 \h  Figure 9-1 . Other HPLC columns,
chromatographic conditions, or detectors may be used if the requirements
for the minimum level (Section   REF _Ref238902775 \r \h  9.7.3 ) and
initial precision and
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Inject 20 μL of the sample extract, Method 1662 extract, or diluted QC
extract into the HPLC using a high-pressure syringe or a constant-volume
sample-injection loop. Record the volume injected to the nearest 0.1
μL.

Upon injection, begin the solvent program used in calibrating the column
(Section   REF _Ref238906903 \r \h  9.7.2.3 ). Record the signal from
the time of injection until the detector returns to a stable baseline.
Return the solvent to the initial conditions.

Using the retention-time data determined during calibration, integrate
the area from the retention time of naphthalene (including the leading
edge of the naphthalene peak) through the end of the
indeno[1,2,3-cd]pyrene peak or until the detector signal returns to a
stable baseline, whichever comes later.

If the height of the response during the period recorded (Section   REF
_Ref238906924 \r \h  9.11.3.2 ) exceeds the height of the response for
phenanthrene during calibration (Section   REF _Ref238906939 \r \h 
9.7.4.2 ), dilute the extract by successive factors of 10 with
acetonitrile and reanalyze until the response is within the calibration
range.

HPLC System and Laboratory Performance

At the beginning of each 8-hour shift during which analyses are
performed, HPLC calibration and system performance are verified. For
these tests, analysis of the medium level calibration standard (  REF
_Ref238900719 \h  Table 9-2 ) and of the diluted extract of the
precision and recovery standard (Section   REF _Ref238901381 \r \h 
9.6.2.3 ) shall be used to verify all performance criteria. Adjustment
and/or recalibration (per Section   REF _Ref238906969 \r \h  9.7 ) shall
be performed until all performance criteria are met. Only after all
performance criteria are met may samples and blanks be analyzed.

Inject 20 μL of the medium-level calibration standard (  REF
_Ref238900719 \h  Table 9-2 ) into the HPLC instrument according to the
procedure in Section   REF _Ref238907025 \r \h  9.11 .

Retention time: The absolute retention times of the naphthalene,
phenanthrene, and indeno[1,2,3-cd]pyrene peaks shall be within ±30
seconds of the respective retention times in the initial calibration
(Section   REF _Ref238906903 \r \h  9.7.2.3 ).

HPLC resolution: Resolution is acceptable if the peak width at
half-height of the phenanthrene peak is less than 30 seconds.

Calibration verification: Compute the concentration of phenanthrene
based on the average calibration factor (Section   REF _Ref238907052 \r
\h  9.7.4.4 ). The concentration shall be within the limits in   REF
_Ref238902725 \h  Table 9-1 . If calibration is verified, system
performance is acceptable and analysis of blanks and QC samples may
begin. If, however, the concentration falls outside of the calibration
verification range, system performance is unacceptable. In this case,
correct the problem and repeat the test, or recalibrate (Section   REF
_Ref238905919 \r \h  9.7.4 ).

Ongoing precision and recovery (OPR): If the extract is from Method
1662, the OPR standard from that method shall be used and the
specification for the OPR from Method 1662 in   REF _Ref238902725 \h 
Table 9-1  shall be met; if not, a sample of diesel oil shall be diluted
per the procedure in Section   REF _Ref238907090 \r \h  9.10  and shall
be used for the OPR test.

Analyze the appropriate OPR standard.

Compute the concentration of PAH in this standard per Section   REF
_Ref238907100 \r \h  9.14 .

Compare the concentration with the limits for ongoing precision and
recovery in   REF _Ref238902725 \h  Table 9-1 . If the concentration is
in the range specified, the analytical processes are in control and
analysis of blanks and samples may proceed. If, however, the
concentration is not in the specified range, these processes are not in
control. In this event, correct the problem, re-extract the sample batch
if the OPR is from Method 1662, or redilute the oil sample (per Section 
 REF _Ref238907127 \r \h  9.10.3 ). and repeat the ongoing precision and
recovery test.

Add results which pass the specification in Section   REF _Ref238906537
\r \h  9.12.6.3  to initial and previous ongoing data. Update QC charts
to form a graphic representation of continued laboratory performance.
Develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation
of percent recovery (Sr). Express the accuracy as a recovery interval
from R – 2Sr, to R + 2 Sr. For example, if R = 95% and Sr = 5%, the
accuracy is 85 to 105%.

Qualitative Identification

Qualitative determination is accomplished by comparison of data from
analysis of a sample or blank with data from analysis of the calibration
verification standard (Section   REF _Ref238901565 \r \h  9.12.5 ).

PAH is identified in the sample by the presence of peaks and/or an
elevated baseline (hump) between the retention times of the naphthalene
and indeno[1,2,3-cd]pyrene peaks (Section   REF _Ref238907351 \r \h 
9.11.3.3 ), as shown in   REF _Ref238901607 \h  Figure 9-1 . The
experience of the analyst shall weigh heavily in interpretation of the
chromatogram.

Quantitative Determination

Using the data system, compute the concentration of the PAH detected in
the solution injected into the HPLC (in μg/mL) using the calibration
factor (Section   REF _Ref238905919 \r \h  9.7.4 ).

Concentration of PAH in oil: If neat oil was analyzed, the concentration
of PAH in the oil is determined using the following equation:

 	[  STYLEREF 1 \s  9 -  SEQ Equation \* ARABIC \s 1  2 ]

Where:

Co 	=	Concentration of PAH in the oil sample

Cp 	= 	Concentration of PAH measured (from Sections   REF _Ref238907387
\r \h  9.11.4  and   REF _Ref238907401 \r \h  9.14.1 )

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Concentration of diesel oil in QC extracts from Method 1662: Calculate
the concentration of diesel oil in QC extracts from Method 1662 by
integrating the area per Section   REF _Ref238901636 \r \h  9.7.6.1  and
using the calibration from Section   REF _Ref238907484 \r \h  9.7.6.2 
of this method, taking into account the dilution of these extracts
(Section   REF _Ref238907498 \r \h  9.10.2 ).

Concentration of PAH in oil from Method 1662: The PAH content of oil is
complicated by the splitting and possible dilution of these extracts.

Concentration in undiluted extracts: This concentration is determined by
Equation 9-3:

   SHAPE  \* MERGEFORMAT   	[  STYLEREF 1 \s  9 -  SEQ Equation \*
ARABIC \s 1  3 ]

Where:

Co 	= 	Concentration of PAH in the oil sample

Ve 	= 	Amount of extract split for HPLC analysis, in mL (1.0 mL)

Cp 	= 	Concentration of PAH measured

WT 	= 	Weight of oil in the concentration tube in Method 1662 (Section
11.5.5 of Method 1662)

1/5 	= 	Fraction of this weight used for the PAH determination

Concentration in diluted extracts: If the extract was diluted by a
factor of 10 (Section   REF _Ref238907127 \r \h  9.10.3  or   REF
_Ref238907387 \r \h  9.11.4 ), the concentration determined in Section  
REF _Ref238907541 \r \h  9.14.4.1  is multiplied by 10.

If the concentration is to be expressed as weight percent, Co is
multiplied by 0.1.

Report results to three significant figures without correction for
recovery.

Method Performance

This method was validated in a single laboratory (Reference 6) using
samples of hot-rolled drilling mud (Reference 7).

References

“Carcinogens-Working With Carcinogens.” Department of Health,
Education, and Welfare, Public Health Service, Centers for Disease
Control [available through National Technical Information System, 5285
Port Royal Road, Springfield, VA 22161, document no. PB2772561: August
1977.

“OSHA Safety and Health Standards, General Industry [29 CFR 1910],
Revised.” Occupational Safety and Health Administration, OSHA 2206.
Washington, DC: January 1976.

“Safety in Academic Chemistry Laboratories (3rd Edition).” American
Chemical Society Publication, Committee on Chemical Safety. Washington,
DC: 1979.

“Handbook of Analytical Quality Control in Water and Wastewater
Laboratories.” USEPA, EMSL-Ci, EPA-600/4-79-019. Cincinnati, OH: March
1979.

“Standard Practice for Sampling Water,” ASTM Annual Book of
Standards, Part 31, D337076, ASTM. Philadelphia, PA: 1980.

“Determination of Polynuclear Aromatic Hydrocarbons and Diesel by
Modified EPA Method 9310.” Prepared for the American Petroleum
Institute c/o Shell Development Co, Westhollow Research Center, 3333
Highway 6 South, Houston, TX 77082 by Analytical Technologies Inc., 225
Commerce Drive, Fort Collins, CO 80524: March 29 1991, April 12, 1991,
and August 18, 1992.

“Results of the API Study of Extraction and Analysis Procedures for
the Determination of Diesel Oil in Drilling Muds (Final Report).”
American Petroleum Institute, Offshore Effluent Guidelines Steering
Committee, Technology Work Group, Prepared by I.C. Raia, Shell
Development Co. Houston, TX: April 18, 1991.

Table   STYLEREF 1 \s  9 -  SEQ Table \* ARABIC \s 1  1 . Performance
Data and Method Acceptance Criteria for PAH

μg/mL	100	—	0.1

Method Detection Limit d	μg/mg	7.6	—	—

Initial prec and recov

Precision (std dev)





PAH in diesel oil e	mg/mL	120	—	—

Diesel in mud extract f	mg/mL	—	0.55	—

Recovery





PAH in diesel oil e	mg/mL	1,090 – 1,340	—	—

Diesel in mud extract f	mg/mL	—	0.84 – 1.95	—

Calibration verification g	μg/mL	—	—	0.39 – 0.61

Ongoing prec and recov

PAH in diesel oil e	mg/mL	1,010 – 1,450	—	—

Diesel in mud extract f	mg/mL	—	0.76 – 2.15	—

Matrix spike recovery f	pct	—	0.43 – 2.39	—

Duplicates	RPD	9.5	44	—

a CAS Registry number 68534-30-5; No. 2 diesel oil used for these tests.

b From Method 1662.

c This is a minimum level at which the analytical system shall give
recognizable signals and acceptable calibration points..

d 40 CFR Part 136, Appendix B; MDL is measured as PAH in oil.

μg/mL.

f Test concentration in diluted extract = 1.25 mg/mL.

g Test concentration = 0.50 μg/mL.

Table   STYLEREF 1 \s  9 -  SEQ Table \* ARABIC \s 1  2 . HPLC
Calibration Data

Analyte	Retention Time a 

(minutes)	Calibration Solution Concentration (μg/mL)



Low	Medium	High

Naphthalene	7.6	—	5	—

Phenanthrene	10.3	0.1	0.5	2.0

Indeno[l23-cd]pyrene	18.9	—	0.5	—

Diesel oil b	7.4 – 20.0	100	400	2,000

a Column system: Two C18 columns (150 mm long by 4.6 mm i.d., 300
angstroms) connected in series,

preceded by one C18 guard column (30 mm long by 4.6 mm i.d., 300
angstroms). Column temperature 30°C;

solvent flow rate 1.5 mL/min; linear gradient from 50% water/50%
acetonitrile at injection to 100%

acetonitrile in 10 minutes, hold at 100% acetonitrile for 15 minutes.

b Diesel oil is calibrated separately using a single point calibration
(Section   REF _Ref239495932 \r \h  9.7.6 ).

Figure   STYLEREF 1 \s  9 -  SEQ Figure \* ARABIC \s 1  1 . Liquid
Chromatography of the Three-Component Standard and of No. 2 Diesel Oil

 PAGE   

CONTENTS (Continued)

Page

 PAGE   iii 

 PAGE   i 

LIST OF TABLES (Continued)

Page

 PAGE   iii 

 PAGE   iv 

LIST OF FIGURES (Continued)

Page

 PAGE   iv 

 PAGE   v 

 PAGE   1-9 

 PAGE   9-15 

2,000 mg carbon	×	240 g	= 480 mg carbon

Per kg dry sediment

1,000

	

Conc. Desired (mg/kg)	×	Dry Weight Sediment (kg)	=	Base Fluid Required
(mg)



g wet sediment	= mL wet sediment

Density (g/mL) of wet sediment

	

Volume sea water per bottle (Eq. 5-9)	= Ratio of sea water:wet sediment

Volume sediment water per bottle (Eq. 5-8)

	

Mean Wet Sediment Weight (g)	= Wet Sediment Density (g/mL)

Mean Wet Sediment Volume (mL)

	

Mean Wet Sediment Weight (g)	= Wet to Dry Ratio

Mean Dry Sediment Weight (g)

	

Conc. Desired (mL/kg)	×	Dry Weight Sediment (kg)	×	Test Substance
Density (g/mL)	=	Test Substance Required (g)



Wet Sediment per Concentration (g)	×	1 kg	= Dry Weight Sediment (kg)

Mean Wet to Dry Ratio

1,000 g

	

Wet Sediment Density (g/mL)	×	Volume of Sediment Required per
Concentration (mL)	=	Weight Wet Sediment Required per Conc. (g)



Mean Wet Sediment Weight (g)	= Wet Sediment Density (g/mL)

Mean Wet Sediment Volume (mL)

	

Wet Sediment Weight (g)	= Wet to Dry Ratio

Dry Sediment Weight (g)

	

 Integrate peaks on the EIP for comparison to calibration to determine
approximate crude oil contamination 

Section 6.11.4.2

Peaks present for Target Aromatics on EIP

 Crude oil contamination is below detection limit.  

Report not detected

Section 6.11.3.3

 Determine the C8 to C13 TIC Area for comparison to calibration
standards 

Section 6.11.2.8

 Obtain the EIP for

m/z 91, m/z 105, and m/z 156

Section 6.11.2.6

C8 to C13 peaks seen on TIC

GC/MS Analyses 

Obtain the TIC for the Sample

Section 6.11.2

Vwell (barrels) = Σ	Bit diameter (in)2	 × change in measured depth
(ft)

	1,029

	

XSVD =	FSVD

	Gwell+ FSVD



Prepare Sample for Analyses

Section 6.11.1

Xi = 	Fi

	G



%BFi =	MBF	 × 100

	Mw

	

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RPD =	D1 – D2	× 100

	(D1 – D2) ÷ 2

	

Co =	Ve × Cp	=	5 × I Cp

	1/5 × WT

WT



Co (mg/g) =	Cp (μg/mL)

	Ci (mg/mL)



RPD =	D1 – D2	 × 100

	(D1 + D2) ÷ 2

	

12 mole CH4	×	22.4 L	×	1,000 ml	×	1 mole hexadecene	×	23 g
hexadecene	×	0.03 kg	= 84 (ml)

mole hexadecene

mole CH4

L

224.4 g hexadecene

kg dry soil

culture

	

VCH4 (ml) =	(S + V) ×	P – Pw	×	CH4	×	273



T + 273

100

760



Corrected % CH1 =	% CH4

	1 –	D × 22.4 L/mol



18 g/mol × 1,000



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60 mg carbon per bottle/1,000	= 0.005 moles carbon

12 g/mole

	

V2 =	P1 × V1 × T2

	T1 × P2



mL wet sediment (Eq. 5-11)	×	Sea water:sediment ratio (Eq. 5-10)	=	mL
sea water to add to wet sediment



480 mg carbon	= 621 mg ethyl oleate

(77.3 ÷ 100)

	

