NOAA Technical Memorandum NOS ORCA 130

National Status and Trends Program

for Marine Environmental Quality

Sampling and Analytical Methods of the

National Status and Trends Program

Mussel Watch Project: 1993-1996 Update

Silver Spring, Maryland

March 1998

US Department of Commerce

noaa NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION

Coastal Monitoring and Bioeffects Assessment Division

Office of Ocean Resources Conservation and Assessment

National Ocean Service
Coastal Monitoring and Bioeffects Assessment Division

Office of Ocean Resources Conservation and Assessment

National Ocean Service

National Oceanic and Atmospheric Administration

U.S. Department of Commerce

N/ORCA2, SSMC4

1305 East-West Highway

Silver Spring, MD 20910

Notice

This report has been reviewed by the National Ocean Service of the National Oceanic and

Atmospheric Administration (NOAA) and approved for publication. Such approval does

not signify that the contents of this report necessarily represent the official position of

NOAA or of the Government of the United States, nor does mention of trade names or

commercial products constitute endorsement or recommendation for their use.
NOAA Technical Memorandum NOS ORCA 130

Sampling and Analytical Methods of the

National Status and Trends Program

Mussel Watch Project:

1993-1996 Update

G. G. Lauenstein and A. Y. Cantillo

(Editors)

Silver Spring, Maryland

March 1998

United States National Oceanic and

Department of Commerce Atmospheric Administration National Ocean Service

William M. Daley D. James Baker Nancy Foster

Secretary Under Secretary Assistant Administrator
TABLE OF CONTENTS

LIST OF TABLES..................................................................................................................i

LIST OF FIGURES.................................................................................................................iii

LIST OF ACRONYMS............................................................................................................iv

Introduction

G. G. Lauenstein and A. Y. Cantillo

ABSTRACT.........................................................................................................................1

1. DISCUSSION..................................................................................................................1

2. BIBIOGRAPHY/REFERENCES...........................................................................................11

ANCILLARY METHODS

Dry Weight Determination of Sediments

S. T. Sweet and T. L. Wade

ABSTRACT.........................................................................................................................14

1. INTRODUCTION..............................................................................................................14

2. APPARATUS AND MATERIALS.......................................................................................14

2.1. Equipment...................................................................................................14

2.2. Reagents....................................................................................................14

3. PROCEDURE..................................................................................................................14

4. CALCULATIONS.............................................................................................................15

4.1. Percent dry weight.....................................................................................15

4.2. Relative Percent Difference between duplicates...........................................15

5. QUALITY CONTROL........................................................................................................15

6. CONCLUSIONS...............................................................................................................15

Determination of Percent Dry Weight for Tissues

Y. Qian and T. L. Wade

ABSTRACT.........................................................................................................................16

1. INTRODUCTION..............................................................................................................16

2. APPARATUS AND MATERIALS.......................................................................................16

2.1. Apparatus..................................................................................................16

2.2. Labware.....................................................................................................16

2.3. Solvents and reagents.................................................................................17

3. PROCEDURE..................................................................................................................17

4. CALCULATIONS.............................................................................................................18

4.1. Percent dry weight.....................................................................................18

4.2. Relative percent difference between duplicates...........................................18

5. QUALITY CONTROL........................................................................................................18

6. CONCLUSIONS...............................................................................................................18

Sediment Grain Size Analysis - Gravel, Sand, Silt and Clay

S. T. Sweet, S. Laswell, and T. L. Wade

ABSTRACT.........................................................................................................................19

1. INTRODUCTION..............................................................................................................19

2. SAMPLE COLLECTION, PRESERVATION AND STORAGE.....................................................19

2.1. Sample collection........................................................................................19
2.2. Sample preservation and storage................................................................19

3. APPARATUS AND MATERIALS.......................................................................................19

3.1. Labware and apparatus...............................................................................19

3.2. Reagents....................................................................................................20

4. PROCEDURE..................................................................................................................20

4.1. Preparation of samples for dry-sieving and pipette analysis........................20

4.2. Size analysis of sand/gravel fraction by wet-sieving..................................20

4.3. Silt/clay sized material by settling.............................................................21

5. CALCULATIONS.............................................................................................................21

6. CONCLUSIONS...............................................................................................................21

7. REFERENCE...................................................................................................................22

Total Organic and Carbonate Carbon Content of Sediments

S. T. Sweet and T. L. Wade

ABSTRACT.........................................................................................................................23

1. INTRODUCTION..............................................................................................................23

2. APPARATUS AND MATERIALS.......................................................................................23

2.1 Equipment.........................................................................................................23

2.2 Reagents..........................................................................................................24

3. Procedure....................................................................................................................24

3.1. LECO system preparation............................................................................24

3.2. Total carbon determination.........................................................................24

3.2.1. Sample preparation.........................................................................24

3.2.2. Sample analyses.............................................................................24

3.2.3. Standard analyses..........................................................................25

3.3. Total Organic Carbon determination.............................................................25

3.3.1. Sample preparation.........................................................................25

3.3.2. Sample analyses.............................................................................25

3.4. Total carbonate carbon content...................................................................25

4. STANDARDIZATION AND CALCULATIONS........................................................................25

5. QUALITY CONTROL........................................................................................................26

6. REPORTING AND PERFORMANCE CRITERIA......................................................................26

7. CONCLUSIONS...............................................................................................................26

Determination of Percent Lipid in Tissue

Y. Qian, J. L. Sericano, and T. L. Wade

ABSTRACT.........................................................................................................................27

1. INTRODUCTION..............................................................................................................27

2. APPARATUS AND MATERIALS.......................................................................................27

2.1. Apparatus..................................................................................................27

2.2. Labware.....................................................................................................27

2.3. Solvents and reagents.................................................................................28

3. PROCEDURE..................................................................................................................28

4. CALCULATIONS.............................................................................................................29

4.1. Percent lipid...............................................................................................29

4.2. Relative percent difference (RPD) for duplicate analysis..............................29

5. QUALITY CONTROL........................................................................................................29

6. CONCLUSIONS...............................................................................................................29
TRACE METAL ANALYSES

TERL Trace Element Quantification Techniques

B. J. Taylor and B. J. Presley

ABSTRACT.........................................................................................................................32

1. INTRODUCTION..............................................................................................................32

2. EQUIPMENT AND SUPPLIES............................................................................................32

2.1. Instrumentation..........................................................................................32

2.2. Supplies.....................................................................................................33

2.3. Labware.....................................................................................................33

2.4. Reagents....................................................................................................33

2.5. Matrix modifiers........................................................................................34

2.6. Standards...................................................................................................34

3. SAMPLE TREATMENT....................................................................................................35

3.1. Oyster and mussel tissue............................................................................35

3.1.1. Bivalve shucking.............................................................................35

3.1.2. Bulk homogenizing...........................................................................35

3.1.3. Freeze drying.................................................................................35

3.1.4. Homogenization of dry aliquot.........................................................35

3.1.5. Digestion........................................................................................35

3.1.6. Displacement volume......................................................................36

3.2. Bottom sediment.........................................................................................36

3.2.1. Homogenization...............................................................................36

3.2.2. Freeze drying.................................................................................36

3.2.3. Homogenization of dry aliquot.........................................................36

3.2.4. Digestion........................................................................................36

4. CALIBRATION AND ANALYSIS........................................................................................37

5. CALCULATIONS.............................................................................................................37

5.1. Concentration.............................................................................................38

5.2. Dilution factor............................................................................................38

5.3. Concentration.............................................................................................38

6. CONCLUSION.................................................................................................................39

7. INSTRUMENTAL ANALYSIS............................................................................................39

7.1. Mercury.....................................................................................................40

7.2. Aluminum...................................................................................................41

7.3. Copper.......................................................................................................42

7.4. Iron............................................................................................................43

7.5. Manganese..................................................................................................44

7.6. Zinc............................................................................................................45

7.7. Silver........................................................................................................46

7.8. Arsenic......................................................................................................48

7.9. Cadmium....................................................................................................50

7.10. Chromium...................................................................................................52

7.11. Copper.......................................................................................................54

7.12. Nickel.........................................................................................................56

7.13. Lead...........................................................................................................58

7.14. Selenium....................................................................................................60

7.15. Tin.............................................................................................................62

7.16. Aluminum...................................................................................................64

7.17. Chromium...................................................................................................65

7.18. Iron............................................................................................................66

7.19. Manganese..................................................................................................67

7.20. Arsenic......................................................................................................68
7.21. Chromium...................................................................................................69

7.22. Iron............................................................................................................70

7.23. Selenium....................................................................................................71

7.24. Silver........................................................................................................72

7.25. Zinc............................................................................................................73

Analysis of Marine Sediment and Bivalve Tissue

E. Crecelius, C. Apts, L. Bingler, J. Brandenberger, M. Deuth, S. Kiesser, and R. Sanders

ABSTRACT.........................................................................................................................74

1. INTRODUCTION..............................................................................................................74

2. EQUIPMENT AND SUPPLIES............................................................................................74

2.1. Instrumentation..........................................................................................74

2.2. Supplies.....................................................................................................75

2.3. Labware.....................................................................................................75

2.4. Reagents....................................................................................................76

2.5. Solvents and matrix modifiers....................................................................76

3. SAMPLE TREATMENT....................................................................................................77

3.1. Drying and homogenization..........................................................................77

3.1.1. Sediments......................................................................................77

3.1.2. Tissues..........................................................................................77

3.2. Digestion....................................................................................................77

3.2.1. Sediments......................................................................................77

3.2.2. Tissues..........................................................................................78

4. CALIBRATION...............................................................................................................78

5. SPECTRAL INTERFERENCES............................................................................................78

6. CALCULATIONS.............................................................................................................79

6.1. Graphite furnace and ICP-MS......................................................................79

6.2. Cold vapor atomic absorption......................................................................79

6.3. X-ray fluorescence.....................................................................................79

7. CONCLUSIONS...............................................................................................................80

8. REFERENCES.................................................................................................................80

9. INSTRUMENTAL ANALYSIS............................................................................................81

9.1. Atomic absorption spectrometry.................................................................81

9.1.1. Aluminum.......................................................................................81

9.1.2. Chromium.......................................................................................82

9.1.3. Nickel.............................................................................................83

9.1.4. Selenium........................................................................................84

9.1.5. Silver............................................................................................85

9.1.5.1. Graphite furnace atomic absorption for tissue..................85

9.1.5.2. Graphite furnace atomic absorption for sediment..............86

9.1.6. Cadmium........................................................................................87

9.1.6.1. Graphite furnace atomic absorption for tissue..................87

9.1.6.2. Graphite furnace atomic absorption for sediment..............88

9.1.7. Mercury.........................................................................................89

9.1.8. Lead...............................................................................................90

9.2. Inductively coupled plasma mass spectrometry...........................................91

9.3. X-Ray fluorescence....................................................................................92
ORGANIC ANALYSES

Extraction and Clean-Up of Sediments for Trace Organic Analysis

Y. Qian, J. L. Sericano, and T. L. Wade

ABSTRACT.........................................................................................................................94

1. INTRODUCTION..............................................................................................................94

2. SAMPLE COLLECTION, PRESERVATION AND STORAGE.....................................................94

2.1. Sample collection........................................................................................94

2.2. Sample preservation and storage................................................................94

3. INTERFERENCES............................................................................................................94

4. APPARATUS AND MATERIALS.......................................................................................95

4.1. Labware and apparatus...............................................................................95

4.2. Reagents....................................................................................................95

5. PROCEDURE..................................................................................................................96

5.1. Sample Preparation....................................................................................96

5.2. Silica/alumina column cleanup.....................................................................96

6. QUALITY CONTROL........................................................................................................97

7. CONCLUSIONS...............................................................................................................97

Extraction of Biological Tissues for Trace Organic Analysis

Y. Qian, J. L. Sericano, and T. L. Wade

ABSTRACT.........................................................................................................................98

1. INTRODUCTION..............................................................................................................98

2. APPARATUS AND MATERIALS.......................................................................................98

2.1. Apparatus..................................................................................................98

2.2. Labware.....................................................................................................98

2.3. Solvents and reagents.................................................................................99

3. PROCEDURE..................................................................................................................99

4. ALUMINA/SILICA GEL CHROMATOGRAPHY.....................................................................101

5. QUALITY CONTROL........................................................................................................101

6. CONCLUSIONS...............................................................................................................101

Procedures for the Extraction of Tissues and Purification of Extracts for

Analysis of Polychlorinated Dibenzo- p-dioxins and Polychlorinated

Dibenzofurans

P. Gardinali, L. Chambers, J. L. Sericano, and T. L. Wade

ABSTRACT.........................................................................................................................102

1. PURPOSE......................................................................................................................102

2. QUALITY CONTROL REQUIREMENTS................................................................................104

2.1. Method blank..............................................................................................104

2.2. Laboratory blank spike................................................................................104

2.3. Ongoing precision and recovery...................................................................104

2.4. Matrix spike...............................................................................................105

2.5. Duplicate....................................................................................................105

2.6. Reference materials...................................................................................105

2.7. Labeled compound recovery........................................................................105

3. APPARATUS AND MATERIALS.......................................................................................105

3.1. Glassware and hardware.............................................................................105

3.2. Reagents and consumable materials.............................................................106

4.2. Analytical standards...................................................................................107

4.2.1. Labeled compound spiking solution...................................................107
4.2.2. Cleanup recovery standard.............................................................107

4.2.3. Precision and recovery standard.....................................................107

4.2.4. Internal standard solution...............................................................109

4.3. Reference materials...................................................................................109

4.3.1. CARP-1..........................................................................................109

4.3.2. EDF-2524.......................................................................................109

4.3.3. EDF-2525.......................................................................................109

4.3.4. EDF-2526.......................................................................................109

4.4. Miscellaneous materials..............................................................................109

5. EXTRACTION AND CLEANUP PROCEDURES......................................................................110

5.1. Sample Preparation....................................................................................110

5.2. Extraction procedure..................................................................................110

5.3. Addition of cleanup recovery standard........................................................110

5.4. Solvent exchange to hexane........................................................................110

5.5. Sulfuric acid/silica gel slurry.....................................................................111

5.6. Rotary evaporation.....................................................................................111

5.7. Mixed bed silica columns.............................................................................111

5.8. Basic alumina column..................................................................................112

5.9. Charcoal column.........................................................................................112

5.10. Final evaporation........................................................................................113

6. CONCLUSIONS...............................................................................................................114

Extraction of Sediments for Butyltin Analysis

Y. Qian, J. L. Sericano, and T. L. Wade

ABSTRACT.........................................................................................................................115

1. INTRODUCTION..............................................................................................................115

2. SAMPLE COLLECTION, PRESERVATION, AND STORAGE....................................................115

3. INTERFERENCES............................................................................................................115

4. APPARATUS AND MATERIALS.......................................................................................116

4.1. Labware and apparatus...............................................................................116

4.2. Reagents....................................................................................................116

5. PROCEDURE..................................................................................................................117

5.1. Sample extraction......................................................................................117

5.2. Hexylation..................................................................................................117

5.3. Silica gel/alumina column cleanup...............................................................118

6. QUALITY CONTROL........................................................................................................118

7. CONCLUSIONS...............................................................................................................119

Extraction of Tissues for Butyltin Analysis

Y. Qian, J. L. Sericano, and T. L. Wade

ABSTRACT.........................................................................................................................120

1. INTRODUCTION..............................................................................................................120

2. SAMPLE COLLECTION, PRESERVATION AND STORAGE.....................................................120

3. INTERFERENCES............................................................................................................120

4. APPARATUS AND MATERIALS.......................................................................................121

4.1. Labware and apparatus...............................................................................121

4.2. Solvents and reagents.................................................................................121

5. PROCEDURES................................................................................................................122

5.1. Preparation of samples...............................................................................122

5.2. Digestion and extraction.............................................................................122

5.3. Hexylation..................................................................................................122

5.4. Silica gel/alumina column cleanup...............................................................123
6. QUALITY CONTROL........................................................................................................123

7. CONCLUSIONS...............................................................................................................124

Purification of Biological Tissue Samples by Gel Permeation Chromatography

for Organic Analyses

Y. Qian, J. L. Sericano, and T. L. Wade

ABSTRACT.........................................................................................................................125

1. INTRODUCTION..............................................................................................................125

2. APPARATUS AND MATERIALS.......................................................................................125

2.1. Equipment...................................................................................................125

2.2. Materials...................................................................................................126

2.3. HPLC calibration standard...........................................................................126

2.4. GPC/HPLC calibration standard...................................................................126

2.5. GPC/HPLC Calibration.................................................................................126

2.6. Preparation of sample extracts for GPC/HPLC............................................127

3. CONCLUSIONS...............................................................................................................128

Quantitative Determination of Polynuclear Aromatic Hydrocarbons by Gas

Chromatography/Mass Spectrometry (GC/MS) - Selected Ion Monitoring (SIM)

Mode

G. J. Denoux, P. Gardinali, and T. L. Wade

ABSTRACT.........................................................................................................................129

1. INTRODUCTION..............................................................................................................129

2. APPARATUS AND MATERIALS.......................................................................................129

3. REAGENTS....................................................................................................................129

3.1. Surrogate spiking solution...........................................................................129

3.2. Internal standard solutions..........................................................................130

3.3. Matrix recovery standard spiking solution..................................................130

3.4. Reference oil solution.................................................................................130

4. GC/MS CALIBRATIONS..................................................................................................130

5. DAILY GC/MS PERFORMANCE TESTS.............................................................................133

6. GAS CHROMATOGRAPHY/MASS SPECTROMETRY Analyses............................................133

7. CALCULATIONS.............................................................................................................134

7.1. Qualitative identification.............................................................................134

7.2. Quantitation................................................................................................134

8. Quality CONTROL/Quality ASSURANCE (QA/QC) REQUIREMENTS.....................................136

8.1. GC/MS tuning.............................................................................................136

8.2. GC/MS initial calibration and continuing calibration checks..........................136

8.3. Standard reference oil................................................................................137

8.4. Method blank analysis.................................................................................137

8.5. Surrogate compound analysis......................................................................137

8.6. Matrix spike analysis.................................................................................138

8.7. Standard Reference Material.......................................................................138

8.8. Method detection limit................................................................................138

9. CONCLUSIONS...............................................................................................................139
Quantitative Determination of Tetra- Through Octa-Polychlorinated Dibenzo- pdioxins and Dibenzofurans by Isotope Dilution High Resolution Gas

Chromatography/High Resolution Mass Spectrometry

L. Chambers, P. Gardinali, J. L. Sericano, and T. L. Wade

ABSTRACT.........................................................................................................................140

1. PURPOSE AND SUMMARY..............................................................................................140

2. QUALITY CONTROL........................................................................................................144

2.1. Instrument criteria.....................................................................................144

2.1.1. Mass spectrometer performance.....................................................144

2.1.2. GC column performance..................................................................144

2.2. Analyte identification criteria.....................................................................147

2.2.1. Retention times..............................................................................147

2.2.2. Ion abundance ratios.......................................................................148

2.2.3. Signal-to-noise ratio......................................................................148

2.2.4. Polychlorinated diphenylether interferences....................................148

2.3. Calibration criteria.....................................................................................148

2.3.1. Initial calibration............................................................................148

2.3.2. Continuing calibration (VER for EPA Method 1613)...........................148

2.4. Criteria for QC samples in an analytical batch.............................................149

2.4.1. Method blank..................................................................................149

2.4.2. Laboratory blank spike....................................................................150

2.4.3. Matrix spike and matrix spike duplicate..........................................150

2.4.4. Duplicate........................................................................................150

2.4.5. Reference material.........................................................................150

2.4.6. Labeled compound recovery............................................................151

3. CHROMATOGRAPHIC CONDITIONS..................................................................................151

3.1. Gas chromatograph.....................................................................................151

3.2. GC columns.................................................................................................151

3.3. Operating conditions...................................................................................151

4. DETECTOR AND DATA SYSTEM CRITERIA.......................................................................152

5. INSTRUMENT CALIBRATION PROCEDURE.........................................................................152

5.1. Initial calibration........................................................................................152

5.2. Continuing calibration verification...............................................................154

6. ANALYTICAL STANDARDS.............................................................................................155

6.1. Labeled compound spiking solution...............................................................155

6.2. Precision and recovery standard.................................................................155

6.3. Internal standard........................................................................................155

6.4. Instrument calibration standards.................................................................155

6.5. Column performance and window defining mix.............................................155

6.6. 2,3,7,8-TCDF CPSM...................................................................................156

7. SAMPLE ANALYSIS.......................................................................................................156

7.1. Qualitative identification.............................................................................156

7.2. Quantitative determination..........................................................................156

7.3. Confirmation analysis.................................................................................157

8. INSTRUMENT MAINTENANCE..........................................................................................157

8.1. Gas chromatograph maintenance..................................................................157

8.2. Mass spectrometer maintenance.................................................................157

9. CONCLUSIONS...............................................................................................................158

10. REFERENCES................................................................................................................158
Quantitative Determination of Chlorinated Hydrocarbons

J. L. Sericano, P. Gardinali, and T. L. Wade

ABSTRACT.........................................................................................................................160

1. INTRODUCTION..............................................................................................................160

2. APPARATUS AND MATERIALS.......................................................................................160

2.1. GC Column..................................................................................................160

2.2. Autosampler...............................................................................................160

3. REAGENTS....................................................................................................................160

3.1. Calibration Solution....................................................................................160

3.2. Surrogate spiking solution...........................................................................161

3.3. Internal standard solution...........................................................................162

3.4. Matrix recovery spiking solution................................................................162

3.5. Retention index solution..............................................................................162

4. PROCEDURE..................................................................................................................162

4.1. Sample extraction and purification..............................................................162

4.2. High resolution GC-ECD analysis..................................................................162

4.2.1. GC conditions.............................................................................162

4.2.2. Calibration.................................................................................163

4.2.3. Sample Analysis.........................................................................163

4.2.4. Calculations...............................................................................163

5. Quality ASSURANCE/Quality CONTROL (QA/QC) REQUIREMENTS.....................................164

5.1. Calibration checks......................................................................................164

5.2. Method blank analysis.................................................................................164

5.3. Surrogate standard analysis.......................................................................165

5.4. Matrix spike/duplicate analysis..................................................................165

5.5. Method detection limit................................................................................165

5.6. GC resolution..............................................................................................165

5.7. Reference material analysis........................................................................166

6. CALCULATIONS.............................................................................................................166

6.1. Chlorinated hydrocarbon calculations..........................................................166

6.2. Calculation notes........................................................................................166

7. REPORTING...................................................................................................................166

8. CONCLUSIONS...............................................................................................................167

9. REFERENCE...................................................................................................................167

Quantitative Determination of Butyltins

J. L. Sericano and T. L. Wade

ABSTRACT.........................................................................................................................168

1. INTRODUCTION..............................................................................................................168

2. APPARATUS AND MATERIALS.......................................................................................168

2.1. GC Column..................................................................................................168

2.2. Autosampler...............................................................................................168

3. REAGENTS....................................................................................................................168

3.1. Calibration solution.....................................................................................168

3.2. Surrogate spiking solution...........................................................................169

3.3. Internal standard solution...........................................................................169

3.4. Matrix recovery spiking solution................................................................169

3.5. Retention Index Solution..............................................................................169

4. PROCEDURE..................................................................................................................169

4.1. Sample extraction and purification..............................................................169

4.2. High resolution GC-FPD analysis..................................................................169

4.2.1. GC conditions...............................................................................169
4.2.2. Calibration..................................................................................170

4.2.3. Retention time windows...............................................................170

4.2.4. Sample analysis...........................................................................170

4.2.5. Calculations.................................................................................171

5. Quality ASSURANCE/Quality CONTROL (QA/QC) REQUIREMENTS.....................................171

5.1. Initial calibration and continuing calibration checks......................................171

5.2. Method blank analysis.................................................................................172

5.3. Surrogate compound analysis......................................................................172

5.4. Matrix spike analysis.................................................................................173

5.5. Method detection limit................................................................................173

5.6. GC resolution..............................................................................................173

5.7. Reference sample analysis..........................................................................173

6. CALCULATIONS.............................................................................................................173

6.1. Butyltin calculations...................................................................................173

6.2. Calculation notes........................................................................................173

7. REPORTING...................................................................................................................174

8. CONCLUSIONS...............................................................................................................174

Battelle Duxbury Operations Trace Organic Analytical Procedures for the

Processing and Analysis of Tissue Samples

C. S. Peven McCarthy and A. D. Uhler

ABSTRACT.........................................................................................................................175

1. INTRODUCTION..............................................................................................................175

2. EQUIPMENT AND REAGENTS...........................................................................................175

2.1. Sample processing equipment and apparatus................................................175

2.2. Reagents....................................................................................................176

2.3. Standard solutions......................................................................................177

3. SUMMARY OF PROCESSING PROCEDURES.......................................................................177

3.1. Initial extraction........................................................................................177

3.2. HPLC cleanup..............................................................................................177

4. SUMMARY OF ANALYSIS METHODS................................................................................178

4.1. Calibration.................................................................................................178

4.2. GC/ECD......................................................................................................178

4.3. GC/MS.......................................................................................................178

5. CALCULATIONS.............................................................................................................179

6. CONCLUSION.................................................................................................................179

7. REFERENCES.................................................................................................................180

NIST Methods for the Certification of SRM 1941a and SRM 1974a

M. M. Schantz, B. A. Benner, Jr., M. K. Donais, M. J. Hays, W. R. Kelly, R. M. Parris, B. J.

Porter, D. L. Poster, L. C. Sander, K. S. Sharpless, R. D. Vocke, Jr., S. A. Wise, M. Levenson,

S. B. Schiller, and M. Vangel

ABSTRACT.........................................................................................................................181

1. INTRODUCTION..............................................................................................................181

2. SRM 1941a, ORGANICS IN MARINE SEDIMENT................................................................182

2.1. Summary...................................................................................................182

2.2. Collection and preparation...........................................................................182

2.3. Moisture determination...............................................................................182

2.4. Polycyclic aromatic hydrocarbons..............................................................183

2.5. Polychlorinated biphenyl congeners and chlorinated pesticides.....................184

2.6. Certified and noncertified concentrations....................................................184

3. SRM 1974a, ORGANICS IN MUSSEL TISSUE (Mytilus edulis)...........................................184
3.1. Summary...................................................................................................184

3.2. Collection and preparation...........................................................................187

3.3. Moisture determination...............................................................................189

3.4. Polycyclic aromatic hydrocarbons..............................................................190

3.5. Polychlorinated biphenyl congeners and chlorinated pesticides.....................191

3.6. Methylmercury..........................................................................................192

3.7. Certified and noncertified concentrations....................................................192

4. CONCLUSION.................................................................................................................193

5. ACKNOWLEDGMENTS AND DISCLAIMER..........................................................................193

6. REFERENCES.................................................................................................................193

HISTOPATHOLOGY

Histopathology Analysis

M. S. Ellis, R. D. Barber, R. E. Hillman, Y. Kim and E. N. Powell

ABSTRACT.........................................................................................................................198

1. INTRODUCTION..............................................................................................................198

2. EQUIPMENT, REAGENTS, SOLUTIONS, AND SAMPLE PREPARATION.................................199

3. ANALYSIS....................................................................................................................199

3.1. Quantitative categories...............................................................................200

3.2. Semi-quantitative categories......................................................................203

4. CONCLUSION.................................................................................................................210

5. REFERENCES.................................................................................................................210

Gonadal Analysis

M. S. Ellis, R. D. Barber, R. E. Hillman, and E. N. Powell

ABSTRACT.........................................................................................................................216

1. INTRODUCTION..............................................................................................................216

2. EQUIPMENT, REAGENTS AND SOLUTIONS........................................................................217

2.1. Equipment...................................................................................................217

2.2. Reagents....................................................................................................218

2.3. Solutions....................................................................................................218

3. SAMPLE COLLECTION AND FIXATION..............................................................................219

3.1. Sampling....................................................................................................219

3.2. Tissue preparation......................................................................................220

3.2.1. Oyster tissue preparation............................................................220

3.2.2. Mussel tissue preparation............................................................221

3.2.3. Zebra mussel preparation.............................................................221

4. SLIDE PREPARATION.....................................................................................................222

4.1. Tissue embedding........................................................................................222

4.2. Tissue sectioning........................................................................................223

4.3. Tissue staining...........................................................................................223

5. ANALYSIS....................................................................................................................223

6. CONCLUSIONS...............................................................................................................225

7. REFERENCES.................................................................................................................226

Perkinsus marinus Assay

E. N. Powell and M. S. Ellis

ABSTRACT.........................................................................................................................228

1. INTRODUCTION..............................................................................................................228

2. EQUIPMENT AND SUPPLIES............................................................................................228
2.1. Reagents....................................................................................................228

2.1.1. Chemicals....................................................................................228

2.1.2. Solutions.....................................................................................229

2.1.2.1. Thioglycollate medium preparation................................229

2.1.2.2. Antibiotic solution........................................................229

2.1.2.3. Lugol's iodine solution...................................................229

2.1.2.4. PBS(II).........................................................................229

2.2. Equipment...................................................................................................229

3. TISSUE COLLECTION......................................................................................................230

4. TISSUE ANALYSIS.........................................................................................................230

4.1. Semiquantitative method.............................................................................230

4.2. Quantitative method....................................................................................231

5. CALCULATIONS.............................................................................................................232

6. CONCLUSIONS...............................................................................................................233

7. REFERENCES.................................................................................................................233

LIST OF TABLES

Lauenstein and Cantillo

1. Laboratories analyzing National Status and Trends Program Mussel Watch

Project samples for trace organics and trace elements............................................2

2. Organic contaminants, major and trace elements and organometallics

determined as part of the NS&T Program................................................................3

3. Mussel Watch Project East and West Coasts tissue polycyclic aromatic

hydrocarbons, method limits of detection................................................................4

4. Mussel Watch Project East and West Coasts tissue pesticides and PCBs, method

limits of detection..................................................................................................5

5. Mussel Watch Project East and West Coasts tissue organotin, method limits of

detection................................................................................................................5

6. Mussel Watch Project Gulf Coast sediment aromatic hydrocarbons, method

limits of detection..................................................................................................6

7. Mussel Watch Project Gulf Coast sediment pesticides and PCBs, method limits

of detection............................................................................................................6

8. Mussel Watch Project Gulf Coast sediment contemporary pesticides, method

limits of detection..................................................................................................7

9. Mussel Watch Project Gulf Coast tissue aromatic hydrocarbons, method limits

of detection............................................................................................................7

10. Mussel Watch Project Gulf Coast tissue pesticides and PCBs, method limits of

detection................................................................................................................8

11. Mussel Watch Project tissue contemporary pesticides, method limits of

detection ...............................................................................................................9

12. Mussel Watch Project Gulf Coast tissue organotin, method limits of detection .........9

13. Mussel Watch Project East and West Coasts tissue major and trace elements,

method limits of detection.......................................................................................10

14. Mussel Watch Project Gulf Coast sediment major and trace elements, method

limits of detection..................................................................................................10

15. Mussel Watch Project Gulf Coast tissue inorganic method limits of detection............11

Taylor and Presley

1. Elemental quantification techniques by matrix.........................................................39

Gardinali et al.

1. Composition of the labeled compound spiking solution...............................................108

2. Composition of the precision and recovery standard solution....................................108

3. Composition of the internal standard solution...........................................................109

Denoux et al.

1. Target compounds...................................................................................................131

2. Final concentration of PAH matrix spike solution.....................................................132

3. Relative retention times and confidence intervals....................................................133

4. Quantitations, confirmation ions, and relative abundance.........................................135

Chambers et al.

i
1. Chlorinated dibenzo-p-dioxins and dibenzofurans determined by isotope dilution

high resolution gas chromatography/high resolution mass spectrometry..................141

2. Retention time references, quantitation references, relative retention times,

and minimum levels for CDDs and CDFs....................................................................142

3. Concentration of PCDDs and PCDFs in calibration and calibration verification

solutions................................................................................................................143

4. Descriptors, exact m/z's, m/z types, and elemental compositions of the CDDs

and CDFs................................................................................................................145

5. GC retention time window defining solution and isomer specificity test

standard.................................................................................................................147

6. Theoretical ion abundance ratios and QC limits.........................................................149

7. Concentration of stock and spiking solutions containing PCDDs and PCDFs labeled

compounds..............................................................................................................159

Sericano et al.

1. Chlorinated hydrocarbons of interest......................................................................161

Sericano and Wade

1. Sample distribution to meet QA requirements during a typical TBT analysis.............171

Schantz et al.

1. Certified concentrations for selected PAHs in SRM 1941a and SRM 1974a...............185

2. Certified concentrations for selected PCB congeners in SRM 1941a and SRM

1974a....................................................................................................................186

3. Certified concentrations for selected chlorinated pesticides in SRM 1941a and

SRM 1974a............................................................................................................187

4. Noncertified concentrations for selected PAHs in SRM 1941a and SRM 1974a..........188

5. Noncertified concentrations for selected PCB congeners and chlorinated

pesticides in SRM 1941a and SRM 1974a................................................................189

Ellis et al.

1. Semi-quantitative scale for digestive gland atrophy................................................204

2. Semi-quantitative scale for Bucephalus infection.....................................................206

3. Semi-quantitative scale for Haplosporidium nelsoni infection ..................................209

4. Semi-quantitative scale for abnormal gonadal development in mussels.....................211

Ellis et al.

1. Tissue embedding sequence.....................................................................................222

2. Tissue staining sequence.........................................................................................224

3. Oyster development stages.....................................................................................224

4. Mussel and zebra mussel development stages..........................................................225

Powell and Ellis

1. Semiquantitative scale of infection intensity for Perkinsus marinus.........................231

ii
LIST OF FIGURES

Gardinali et al.

1. Flow chart for sample processing............................................................................103

Ellis et al.

1. Unidentified worm in the gonoduct of an oyster, Crassostrea virginica.....................201

2. Edema in the gill of an oyster, Crassostrea virginica...............................................202

3. Edema in the connective tissue of an oyster, Crassostrea virginica..........................203

4. Crassostrea virginica normal digestive tubule, scored a 0 according to

Table 1...................................................................................................................205

5. Digestive gland atrophy in Crassostrea virginica scored a 2 according to

Table 1...................................................................................................................205

6. Digestive gland atrophy in Crassostrea virginica scored a 4 according to

Table 1...................................................................................................................206

7. Bucephalus infection in Mytilus edulis scored a 1 according to Table 2. Some

gametic tissue is still present.................................................................................207

8. Bucephalus infection in Mytilus edulis scored a 2 according to Table 2. No

gametic tissue is present........................................................................................207

9. Bucephalus infection in Mytilus edulis scored a 3 according to Table 2. No

gametic tissue is present. Bucephalus heavily infiltrating digestive gland.................208

10. Mytilus edulis follicle with abnormal gametic tissue................................................209

11. Mytilus edulis follicle heavily infiltrated with hemocytes........................................210

Ellis et al.

1. Oyster tissue used for quantifying reproductive stage.............................................220

iii
LIST OF ACRONYMS

ACS American Chemical Society

Alumina Used as an adsorbent in liquid-solid chromatography made by dehydrating

alumina trihydrate

AMU Atomic Mass Unit

CAS Chemical Abstract Service

CRM Certified Reference Material, prepared by the National Research Council of

Canada

CRS Clean-up Recovery Standard

CVAA Cold vapor atomic absorption spectrometry

DB5 Capillary column, internal coating 5% phenyl silicone and 95% methyl

silicone

DBT Dibutyltin

DDT 1,1,1-Trichloro-2,2-bis[p-chlorophenyl]ethane

ECD Electron capture detector

EDL Electrodeless discharge lamp

EPA U.S. Environmental Protection Agency

FAAS Flame atomic absorption spectrometry

GC Gas Chromatography

GERG Geochemical and Environmental Research Group, Texas A&M University

GFAA Graphite furnace atomic absorption spectrometry

GPC Geopermeation chromatography

HCH 1,2,3,4,5,6-Hexachlorocyclohexane. Lindane is gamma-HCH

HCL Hollow cathode lamp

HEPA High efficiency particulate attenuator

HPLC High performance liquid chromatography

ICP-MS Inductively coupled plasma - mass spectrometry

i.d. Internal diameter

IS Internal standard

IUPAC International Union of Pure and Applied Chemistry

LBS Laboratory Spike Sample

LC-FL Liquid chromatography with fluorescence detection

LCSS Labeled compound spiking solution

MBT Monobutyltin

MDL Method detection limit

ML Minimum level

MS Mass spectrometry in discussions of analytical methodology

Matrix spike in discussions of Quality Assurance/Quality Control

MSD Matrix spike duplicate

NIST National Institute of Standards and Technology

NMFS National Marine Fisheries Service, NOAA

NOAA National Oceanic and Atmospheric Administration, Department of Commerce

NOS National Ocean Service, NOAA

NRC National Research Council (Canada)

Nsec Nanosecond

NS&T National Status and Trends Program

NWFSC NOAA/NMFS/Northwest Fisheries Science Center

OPR Ongoing precision and recovery, sample

PAH Polycyclic aromatic hydrocarbons

PAR Precision and recovery spiking solution

PCB Polychlorinated biphenyl

PFTBA Perfluorotributylanine

iv
PPB Concentrations of parts per billion, ng/g (Mussel Watch Project data are

reported on a dry weight basis)

PPM Concentrations of parts per million, μg/g (Mussel Watch Project data are

reported on a dry weight basis)

PPQ Concentrations of parts per quadrillion, pg/L (for water samples, not

regularly quantified in the Mussel Watch Project)

PPT Concentrations of parts per trillion, pg/g (used to report low level

contaminants such as dioxins and furans on a dry weight basis)

QA Quality assurance

QC Quality control

RM Reference Material

RPD Relative percent difference

RPM Revolutions per minute

SICP Selected ion current profile

SIM Selected ion monitoring

SOP Standard Operating Procedure

SRM Standard reference material, prepared by the National Institute of

Standards and Technology

TAMU Texas A&M University

TERL Trace Element Research Laboratory, Department of Oceanography, Texas

A&M University

TBT Tributyltin

XRF X-ray fluorescence spectrometry

v
vi
Introduction

G. G. Lauenstein and A. Y. Cantillo

(Editors)

Coastal Monitoring and Bioeffects Assessment Division

Office of Ocean Resources Conservation and Assessment

National Ocean Service

National Oceanic and Atmospheric Administration

Silver Spring, MD

ABSTRACT

Polycyclic aromatic hydrocarbons, butyltins, polychlorinated biphenyls, DDT and

metabolites, other chlorinated pesticides, trace and major elements, and a number of

measures of contaminant effects are quantified in bivalves and sediments collected as

part of the NOAA National Status and Trends (NS&T) Program. This document contains

descriptions of some of the sampling and analytical protocols used by NS&T contract

laboratories from 1993 through 1996.

1. DISCUSSION

The quantification of environmental contaminants and their effects by the National Oceanic and

Atmospheric Administration's National Status and Trends (NS&T) Program began in 1984.

Polycyclic aromatic hydrocarbons, butyltins, polychlorinated biphenyls, DDT and metabolites,

other chlorinated pesticides, trace and major elements, and a number of measures of

contaminant effects are quantified in estuarine and coastal samples. There have been two major

monitoring components within this program: the National Benthic Surveillance Project which

was responsible for quantification of contamination in fish tissue and sediments, and developing

and implementing new methods to define the biological significance of environmental

contamination; and the Mussel Watch Project which currently monitors pollutant concentrations

by quantifying contaminants in bivalve mollusks and sediments. Methods for sample collection,

preparation, and quantification through 1992 were described in NOAA Technical Memorandum

NOS ORCA 71 (Vols. 1 - 4). Part of the NS&T Program is the performance-based Quality

Assurance Project which allows for the documentation of methodology and laboratory

performance through time as analytical procedures change. Methods in the following pages are

those used by laboratories that have worked on the NS&T Project since 1992 as well as the

methods from the National Institute of Standards and Technology, the group that oversees the

quality of organic contaminant analyses. Summaries of the methods used by the National

Research Council of Canada, which is responsible for the assurance of quality for trace element

analyses, are available in other NOS ORCA technical memoranda.

Table 1 shows the time periods laboratories were responsible for various aspects of the Mussel

Watch Project (since 1992), and the authors of the following chemistry chapters. Table 2 lists

the major and trace elements, and organic contaminants measured as part of the core Mussel

Watch Project.

Not only have analytical methods used within the Mussel Watch Project changed with time but

the analytes measured have also changed. The core list of trace elements and PAHs has

remained the same but since 1995, the NS&T Project no longer regularly reports PCB 77 and

126. While these two congeners are quantifiable during the analyses for the other 18 PCB

congeners measured by the NS&T Project, PCB 77 and PCB 126 are only a small percentage of

the co-eluting PCBs with which they are associated. Because planar PCBs are of interest to the

NS&T Project, PCBs 77, 126, and 169 have been measured in select samples since 1995. While

1
the core list of PAHs measured by the NS&T Project has remained the same the value of also

measuring alkylated PAHs has became apparent and these compounds have been measured

aperiodically since 1993. Beginning in 1995, furans, dioxins and other water soluble

contemporary pesticides have also been measured at selected sites.

Detection limits do change as a function of analytical techniques used, detection limits for the

year 1993 -1996 for each laboratory participating in the Mussel Watch Project are found in

Tables 3 - 15. For information on the analytical evolution of the NS&T Program (both the

National Benthic Surveillance and Mussel Watch Projects), early analytical methods, and early

detection limits (and how they were derived) see NOAA Technical Memorandum NOS ORCA 71.

Table 1. Laboratories analyzing National Status and Trends Program Mussel Watch Project

samples for trace organics and trace elements.

Trace Organic Analyses............................................................................................................

Year 1992-1994 1995-1996

East Coast* Battelle• TAMU/GERG

(Peven-MacCarthy and Uhler) (Wade et al.)

Gulf Coast TAMU/GERG TAMU/GERG

(Wade et al.) (Wade et al.)

West Coast Battelle TAMU/GERG

(Peven-MacCarthy and Uhler) (Wade et al.)

Trace Element Analyses............................................................................................................

Year 1992-1994 1995-1996

East Coast Battelle TAMU/TERL

(Crecelius et al.) (Taylor and Presley)

Gulf Coast TAMU/TERL TAMU/TERL

(Taylor and Presley) (Taylor and Presley)

West Coast Battelle TAMU/TERL

(Crecelius et al.) (Taylor and Presley)

* East Coast samples include those samples collected in the Great Lakes. Gulf coast sites include those sites collected from

Puerto Rico. West Coast sites include sites in Alaska and Hawaii.

• Battelle - Battelle, Duxbury, MA (trace organic analyses) and Sequim, WA (trace element).

TAMU/GERG - Geochemical and Environmental Research Group of Texas A&M University, College Station, TX.

TAMU/TERL - Trace Element Research Laboratory, Department of Oceanography, Texas A&M University, College Station, TX.

2
Table 2. Organic contaminants, major and trace elements and organometallics determined as

part of the NS&T Program.

Polycyclic aromatic hydrocarbons

Low molecular weight PAHs High molecular weight PAHs

(2- and 3-ring structures) (4-, 5-, and 6-rings)

1-Methylnaphthalene Naphthalene Benz[a]anthracene

1-Methylphenanthrene C1-Naphthalenes Benzo[a]pyrene

2-Methylnaphthalene C Benzo[b]fluoranthene

2

-Naphthalenes

2,6-Dimethylnaphthalene Benzo[e]pyrene C3

-Naphthalenes

1,6,7-Trimethylnaphthalene Benzo[ghi]perylene C4

-Naphthalenes Acenaphthene Benzo[k]fluoranthene

Phenanthrene Acenaphthylene Chrysene

C1-Phenanthrenes/ Anthracene C1

-Chrysenes

Biphenyl Anthracenes C2

-Chrysenes

C2 Dibenzothiophene -Phenanthrenes/ C3

-Chrysenes

C1-Dibenzothiophenes Anthracenes C4

-Chrysenes C3 C -Phenanthrenes/

2

-Dibenzothiophenes Dibenz[a,h]anthracene

C Anthracenes

3

-Dibenzothiophenes Fluoranthene C4

-Phenanthrenes/ Fluorene C1

-Fluoranthenes/Pyrenes

C Anthracenes

1

-Fluorenes Indeno[1,2,3-cd]pyrene

C2-Fluorenes Perylene

C3-Fluorenes Pyrene

Chlorinated pesticides (* - determined since 1995)

2,4'- DDD cis-Chlordane Heptachlor epoxide

4,4'- DDD Dieldrin Hexachlorobenzene

2,4'- DDE Endosulfan-I * alpha-Hexachlorohexane

4,4'- DDE Endosulfan-II * beta-Hexachlorohexane *

2,4'-DDT delta-Hexachlorohexane * Mirex

4,4'- DDT gamma-Chlordane * cis-Nonachlor

Aldrin gamma-Hexachlorohexane * trans-Nonachlor

Chlorpyrifos * Heptachlor Oxychlordane *

Polychlorinated biphenyl congeners (IUPAC numbering system)

PCB 8, PCB 18, PCB 28, PCB 44, PCB 52, PCB 66, PCB 101, PCB 105, PCB 118, PCB 128,

PCB 138, PCB 153, PCB 170, PCB 180, PCB 187, PCB 195, PCB 206, PCB 209

Planar PCBs (PCB 77, PCB 126, PCB 169)

3
Table 2. Organic contaminants, and major and trace elements determined as part of the NS&T

Program (cont.).

Chlorinated dibenzofurans (determined

since 1995)

Chlorinated dioxins (determined since

1995)

2,3,7,8-Tetrachlorodibenzofuran 2,3,7,8-Tetrachlorodibenzo-p-dioxin

1,2,3,7,8-Pentachlorodibenzofuran 1,2,3,7,8-Pentachlorodibenzo-p-dioxin

2,3,4,7,8-Pentachlorodibenzofuran 1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin

1,2,3,4,7,8-Hexachlorodibenzofuran 1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin

1,2,3,6,7,8-Hexachlorodibenzofuran 1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin

2,3,4,6,7,8-Hexachlorodibenzofuran 1,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin

1,2,3,7,8,9-Hexachlorodibenzofuran Octachlorodibenzo-p-dioxin

1,2,3,4,6,7,8-Heptachlorodibenzofuran

1,2,3,4,7,8,9-Heptachlorodibenzofuran

Octachlorodibenzofuran

Major and trace elements

Al, Si, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Sn, Sb, Ag, Cd, Hg, Tl, Pb

Organotins

Monobutyltin3+, dibutyltin2+, tributyltin+, tetrabutyltin

 Only the cation portion of the molecule is quantified because many anions can combine with the tin-containing cation.

Tributyltin is the primary biocide; dibutyltin and monobutyltin are metabolites of tributyltin; and tetrabutyltin is an unintended

manufacturing by-product.

Table 3. Mussel Watch Project East and West Coasts tissue polycyclic aromatic hydrocarbons,

method limits of detection (ng/g dry weight).

Compound 1993 and 1994 Compound 1993 and 1994

Acenaphthene 2.7 2-Methylnaphthalene 2.3

Acenaphthylene 2.1 1-Methylphenanthrene 3.0

Anthracene 2.4 Naphthalene 19

Benz[a]anthracene 1.8 Perylene 6.7

Dibenz[a,h]anthracene 3.8 Phenanthrene 5.5

Benzo[b]fluoranthene 2.1 Pyrene 5.2

Benzo[k]fluoranthene 4.7 1,6,7-Trimethylnaphthalene 1.8

Benzo[ghi]perylene 5.1

Benzo[a]pyrene 5.0 C1 to C4 - Naphthalenes 19

Benzo[e]pyrene 4.0 C1

 to C3 - Fluorenes 2.7

Biphenyl 4.8 C1

 to C4 - Phenanthrenes +

Chrysene 1.7 anthracenes 5.5 2,6-Dimethylnaphthalene 3.6 Dibenzothiophene 2.7 Fluoranthene 5.0 C1

 to C3 - Dibenzothiophenes 2.7 Fluorene 2.7

C1 - Fluoranthenes + pyrenes 5.2 Indeno[1,2,3-cd]pyrene 2.8

C1 to C4 1-Methylnaphthalene 2.6 - Chrysenes 1.7

4
Table 4. Mussel Watch Project East and West Coasts tissue pesticides and PCBs, method limits

of detection (ng/g dry weight).

Compound 1993 1994 Compound 1993 1994

Aldrin 1.8 1.3 PCB 8 3.9 4.7

cis-Chlordane 1.4 1.8 PCB 18 2.2 1.5

Dieldrin 2.3 2.2 PCB 28 1.8 0.85

Heptachlor 3.3 0.85 PCB 44 1.4 1.3

Heptachlor epoxide 1.6 0.75 PCB 52 1.8 1.1

Hexachlorobenzene 1.0 0.74 PCB 66 3.3 0.67

gamma-HCH 1.8 0.92 PCB 101 1.5 0.72

Mirex 2.1 2.1 PCB 105 2.3 0.37

trans-Nonachlor 2.2 0.97 PCB 118 2.6 0.88

2,4'-DDD 4.1 0.89 PCB 128 1.9 1.3

4,4'-DDD 3.2 0.69 PCB 138 2.1 0.76

2,4'-DDE 1.5 0.94 PCB 153 4.5 1.1

4,4'-DDE 2.6 2.5 PCB 170 2.2 2.2

2,4'-DDT 3.1 0.97 PCB 180 2.5 2.1

4,4'-DDT 3.1 1.3 PCB 187 2.5 0.86

PCB 195 1.7 1.2

PCB 206 1.6 1.9

PCB 209 1.9 3.3

Table 5. Mussel Watch Project East and West Coasts tissue organotin, method limits of

detection (ng/g cation, dry weight).

1993 1994

Compound

Monobutyltin (MBT) 12 7.6

Dibutyltin (DBT) 12 7.5

Tributyltin (TBT) 19 7.6

Tetrabutyltin 6.7 7.6

5
Table 6. Mussel Watch Project Gulf Coast (until 1995 and all coasts thereafter) sediment

aromatic hydrocarbons, method limits of detection (ng/g dry weight).

Compound 1993 and 1995 and Compound 1993 and 1995 and

1994 1996 1994 1996

Acenaphthene 0.6 0.5 Perylene 2.7 0.6

Acenaphthylene 0.5 0.3 Phenanthrene 0.6 0.8

Anthracene 0.5 0.5 Pyrene 0.4 1.1

Benz[a]anthracene 0.4 0.2 1,6,7-Trimethyl=

Benzo[b]fluoranthene 0.3 1.3 naphthalene 0.7 0.4

Benzo[k]fluoranthene 0.3 0.5

Benzo[ghi]perylene 3.0 1.3 C1 - Naphthalenes 2.3 2.6

Benzo[a]pyrene 0.3 0.6 C2

 - Naphthalenes 1.2 1.2

Benzo[e]pyrene 0.2 0.6 C3

 to C4 - Naphthalenes 1.3 0.9

Biphenyl 0.4 0.3 C1

 to C3 - Fluorenes 1.2 1.0 Chrysene 0.3 0.7

C1 to C4 - Phenanthrenes Dibenz[a,h]anthracene 0.4 0.5

2,6-Dimethylnaphthalene 0.6 0.6 + anthracenes 1.1 0.5

Fluoranthene 0.3 1.0 Dibenzothiophene 0.5 0.3

C1 to C3 Fluorene 0.6 0.5 - Dibenzo=

Indeno[1,2,3-cd]pyrene 0.3 0.3 thiophenes 1.0 0.6

1-Methylnaphthalene 1.1 1.0 C1 - Fluoranthenes

2-Methylnaphthalene 1.2 1.7 + pyrenes 0.7 2.1

1-Methylphenanthrene 0.5 0.2 C1

 to C4 - Chrysenes 0.7 1.4

Naphthalene 1.1 2.2

Table 7. Mussel Watch Project Gulf Coast (through 1994 and all coasts thereafter) sediment

pesticides and PCBs, method limits of detection (ng/g dry weight).

Compound 1993 - 1995 1996 Compound 1993 - 1995 1996

Aldrin 0.04 0.13 PCB 8 0.14 0.12

cis-Chlordane 0.04 0.08 PCB 18 0.07 0.82

Dieldrin 0.42 0.04 PCB 28 0.06 0.09

Heptachlor 0.19 0.05 PCB 44 0.08 0.10

Heptachlor epoxide 0.32 0.04 PCB 52 0.07 0.42

Hexachlorobenzene 0.05 0.07 PCB 66 0.10 0.07

gamma-HCH 0.04 0.08 PCB 101 0.09 0.15

Mirex 0.04 0.11 PCB 105 0.17 0.06

trans-Nonachlor 0.04 0.10 PCB 118 0.32 0.07

PCB 128 0.22 0.14

2,4'-DDD 0.04 0.18 PCB 138 0.17 0.07

4,4'-DDD 0.11 0.07 PCB 153 0.15 0.08

2,4'-DDE 0.05 0.08 PCB 170 0.12 0.17

4,4'-DDE 0.06 0.06 PCB 180 0.09 0.05

2,4'-DDT 0.33 0.05 PCB 187 0.11 0.08

4,4'-DDT 0.08 0.09 PCB 195 0.11 0.09

PCB 206 0.13 0.05

PCB 209 0.22 0.10

6
Table 8. Mussel Watch Project Gulf Coast (through 1994 and all coasts thereafter) sediment

contemporary pesticides, method limits of detection (ng/g dry weight).

Compound 1995* 1996

1,2,4,5-Tetracholorobenzene - 0.71

1,2,3,4-Tetracholorobenzene - 0.98

Pentachlorobenzene - 0.11

Pentachloroanisole - 0.05

Chlorpyrifos - -

Dicofol - -

alpha-HCH 0.09 0.37

beta-HCH 0.03 0.17

delta-HCH 0.17 0.05

Oxychlordane 0.18 0.07

gamma-Chlordane 0.05 0.15

cis-Nonachlor 0.03 0.04

Endosulfan I - -

Endosulfan II 0.08 0.06

* These additional analytes were first quantified at selected sites on the three major U.S. coasts beginning in 1995.

Table 9. Mussel Watch Project Gulf Coast (through 1994 and all coasts thereafter) tissue

aromatic hydrocarbons, method limits of detection (ng/g dry weight).

Compound 1993 and 1995 and Compound 1993 and 1995 and

1994 1996 1994 1996

Acenaphthene 8.5 12 Perylene 4.8 10.1

Acenaphthylene 7.3 4.8 Phenanthrene 3.5 6.6

Anthracene 2.3 4.5 Pyrene 4.4 5.6

Benz[a]anthracene 5.1 2.5 1,6,7-Trimethyl=

Benzo[b]fluoranthene 4.0 8.7 naphthalene 12 8.4

Benzo[k]fluoranthene 4.0 5.4

Benzo[ghi]perylene 3.0 4.1 C1 - Naphthalenes 20 15

Benzo[a]pyrene 4.9 4.7 C2

 - Naphthalenes 17 21

Benzo[e]pyrene 2.5 4.3 C3

 to C4 - Naphthalenes 25 17

Biphenyl 9.2 10 C1

 to C3 - Fluorenes 19 14 Chrysene 11 5.8

C1 to C4 - Phenanthrenes Dibenz[a,h]anthracene 2.8 6.6

2,6-Dimethylnaphthalene 8.7 10 + anthracenes 30 18

Fluoranthene 4.2 5.8 Dibenzothiophene 2.2 8.7

C1 to C3 Fluorene 9.3 6.8 - Dibenzo=

Indeno[1,2,3-cd]pyrene 5.6 6.5 thiophenes 4.4 17

1-Methylnaphthalene 9.0 6.4 C11 - Fluoranthenes

2-Methylnaphthalene 11 8.5 + pyrenes 8.7 11

1-Methylphenanthrene 15 9.1 C1

 to C4 - Chrysenes 22 12

Naphthalene 6.2 8.3

7
Table 10. Mussel Watch Project Gulf Coast (through 1994 and all coasts thereafter) tissue

pesticides and PCBs, method limits of detection (ng/g dry weight).

Compound 1993 1994 and 1995 1996

Aldrin 0.49 0.41 1.1

cis-Chlordane 0.75 0.49 0.39

Dieldrin 0.66 1.0 1.7

Heptachlor 0.52 0.53 0.54

Heptachlor epoxide 0.57 0.09 0.31

Hexachlorobenzene 0.54 0.40 0.75

gamma-HCH 0.33 0.25 0.77

Mirex 0.54 0.28 0.62

trans-Nonachlor 1.9 0.25 0.35

2,4'-DDD 0.64 0.17 0.37

4,4'-DDD 3.7 0.28 0.92

2,4'-DDE 0.30 0.44 0.35

4,4'-DDE 0.76 0.92 1.3

2,4'-DDT 0.47 0.31 0.53

4,4'-DDT 0.38 0.83 1.1

PCB 8 0.84 1.4 0.82

PCB 18 0.52 1.1 1.0

PCB 28 0.35 0.75 0.88

PCB 44 0.24 0.62 0.75

PCB 52 0.92 1.0 1.1

PCB 66 0.39 0.65 0.45

PCB 101 0.51 0.47 0.47

PCB 105 1.1 1.2 0.55

PCB 118 0.47 1.3 0.96

PCB 128 0.40 0.62 3.6

PCB 138 5.9 1.0 1.1

PCB 153 1.6 2.6 1.1

PCB 170 0.36 2.4 1.1

PCB 180 0.36 0.76 0.53

PCB 187 0.71 0.38 0.62

PCB 195 0.89 0.82 0.69

PCB 206 0.59 0.48 0.67

PCB 209 0.59 1.1 0.77

8
Table 11. Mussel Watch Project tissue contemporary pesticides, method limits of detection

(ng/g dry weight).

Compound 1995* 1996

1,2,4,5-Tetracholorobenzene - 0.83

1,2,3,4-Tetracholorobenzene - 1.7

Pentachlorobenzene - 0.89

Pentachloroanisole - 0.63

Chlorpyrifos - -

Dicofol - -

alpha-HCH 0.09 0.49

beta-HCH 0.03 0.92

delta-HCH 0.17 0.83

Oxychlordane 0.18 0.72

gamma-Chlordane 0.04 0.39

cis-Nonachlor 0.03 0.62

Endosulfan I - -

Endosulfan II 0.08 1.1

* These additional analytes were first quantified at selected sites on the three major U.S. coasts beginning in 1995.

Table 12. Mussel Watch Project Gulf Coast (through 1994 and all coasts thereafter) tissue

organotin, method limits of detection (ng/g Sn, dry weight).

1993 - 1994 1995 - 1996

Compound Bivalves Sediments Bivalves Sediments

Monobutyltin (MBT) 6.5 1.0 8.8 15

Dibutyltin (DBT) 5.6 1.0 8.6 15

Tributyltin (TBT) 5.5 1.0 12 15

Tetrabutyltin 4.7 1.0 11 15

9
Table 13. Mussel Watch Project East and West Coasts tissue major and trace elements, method

limits of detection (μg/g dry weight).

Elements 1993 1994

Al 18 120

Si - 300

Cr 0.58 0.41

Mn 6.7 21

Fe 34 920

Ni 0.79 0.74

Cu 2.2 110

Zn 39 1400

As 2.6 1.8

Se 1 3.7

Ag 0.14 0.42

Cd 0.54 0.33

Sn 0.35 0.60

Sb - 0.090

Hg 0.021 0.076

Tl -

Pb 0.059 0.19

Table 14. Mussel Watch Project Gulf Coast (through 1994 and all coasts thereafter) sediment

major and trace elements, method limits of detection (μg/g dry weight).

Elements 1993 1994 1995 1996

Al 200 200 360 106

Cr 0.05 0.10 0.50 0.64

Mn 3.5 3.5 7.8 2.5

Fe 11 100 480 290

Ni 0.4 0.40 0.33 0.19

Cu 0.44 0.44 0.47 0.30

Zn 1.5 1.5 11 0.78

As 0.24 0.25 0.10 0.31

Se 0.10 0.10 0.01 0.02

Ag 0.013 0.013 0.01 0.011

Cd 0.018 0.018 0.013 0.003

Sn 0.10 0.10 0.093 0.11

Sb 0.10 0.10 0.08 0.15

Hg 0.006 0.01 0.005 0.005

Tl --- --- 0.20 0.04

Pb 0.15 0.15 0.44 0.35

10
Table 15. Mussel Watch Project Gulf Coast (through 1994 and all coasts thereafter) tissue

inorganic method limits of detection (μg/g dry weight).*

Elements 1993 1994 1995 1996

Al 0.70 120 120 10

Cr 0.15 0.50 0.50 0.40

Mn --- 3.8 3.8 1.7

Fe 8.5 490 100 15

Ni 0.24 0.15 0.15 0.14

Cu 0.18 0.2 0.16 0.6

Zn 1.2 2.3 6.5 5.7

As 0.2 0.41 0.40 0.35

Se 0.15 0.60 0.50 0.41

Ag 0.035 0.10 0.10 0.29

Cd 0.012 0.00 0.003 0.008

Sn 0.12 0.14 0.14 0.28

Sb - - 0.08 0.15

Hg 0.016 0.012 0.012 0.015

Tl - - 0.20 0.038

Pb 0.10 0.11 0.11 0.10

2. BIBLIOGRAPHY/REFERENCES

Battelle Ocean Sciences (1993) National Status and Trends Mussel Watch Project Year 8 Final

Report: Collection of Bivalves and Surficial Sediments from Coastal U.S. Atlantic and Pacific

Locations and Analyses for Organic Chemicals and Trace Elements. Battelle Ocean Sciences,

Duxbury, MA. 88 pp. plus appendices

Battelle Ocean Sciences (1994) National Status and Trends Mussel Watch Project Year 9 Final

Report: Collection of Bivalves and Surficial Sediments from Coastal U.S. Atlantic and Pacific

Locations and Analyses for Organic Chemicals and Trace Elements. Battelle Ocean Sciences,

Duxbury, MA. 74 pp. plus appendices.

Geochemical and Environmental Research Group (1994) NOAA National Status and Trends

Mussel Watch Program: Analytical Data Report Year VIII. Geochemical and Environmental

Research Group, Texas A&M University, College Station, TX. 370 pp.

Geochemical and Environmental Research Group (1995) NOAA National Status and Trends

Mussel Watch Program: Analytical Data Report Year IX. Geochemical and Environmental

Research Group, Texas A&M University, College Station, TX. 154 pp.

Lauenstein, G. G., A. Y. Cantillo, S. Kokkinakis, J. Jobling, and R. Fay (1997) Mussel Watch

Project Site Descriptions, through 1997. NOAA Technical Memorandum NOS ORCA 112, Silver

Spring, MD. 354 pp.

11
Lauenstein, G. G., A. Y. Cantillo, and S. S. Dolvin (1993) Sampling and analytical methods of the

National Status and Trends Program National Benthic Surveillance and Mussel Watch Projects

1984-1992: Overview and summary of methods, Volume I. NOAA Technical Memorandum NOS

ORCA 71, Silver Spring, MD. 117 pp.

Lauenstein, G. G. and A. Y. Cantillo (eds.) (1993) Sampling and analytical methods of the

National Status and Trends Program National Benthic Surveillance and Mussel Watch Projects

1984-1992: Comprehensive descriptions of complementary measurements, Volume II. NOAA

Technical Memorandum NOS ORCA 71, Silver Spring, MD. 102 pp.

Lauenstein, G. G. and A. Y. Cantillo (eds.) (1993) Sampling and analytical methods of the

National Status and Trends Program National Benthic Surveillance and Mussel Watch Projects

1984-1992: Comprehensive descriptions of elemental analytical methods, Volume III. NOAA

Technical Memorandum NOS ORCA 71, Silver Spring, MD. 219 pp.

Lauenstein, G. G. and A. Y. Cantillo (eds.) (1993) Sampling and analytical methods of the

National Status and Trends Program National Benthic Surveillance and Mussel Watch Projects

1984-1992: Comprehensive descriptions of trace organic analytical methods, Volume IV. NOAA

Technical Memorandum NOS ORCA 71, Silver Spring, MD. 182 pp.

12
ANCILLARY METHODS

13
Dry Weight Determination of Sediments

S. T. Sweet and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

The percent dry weight determination procedure used for the NOAA National Status and

Trends Mussel Watch Project sediment samples is described. Sediment dry weight was

obtained by drying the samples to a constant weight.

1. INTRODUCTION

Dry weight measurements of sediments are necessary when results of sediment analyses are

expressed on a dry weight basis. Once the dry weight has been determined, the percent

moisture can also be calculated.

2. APPARATUS AND MATERIALS

2.1. Equipment

Balance, analytical, capable of measuring milligram accuracy.

Calibrated weights

Oven, drying capable of maintaining 63 - 65 °C.

Spatula

Vials, scintillation, combusted glass, 20-dram

2.2. Reagents

Methanol (CH3OH) [67-56-1] (pesticide quality or equivalent).

Dichloromethane (CH2Cl2) [75-09-2] (pesticide quality or equivalent.

3. PROCEDURE

Sediments were collected in pre-cleaned and/or pre-combusted glass jars and stored frozen

(-20 °C). Sediments were thawed and homogenized using a solvent rinsed spatula. The spatula

was triple rinsed, first using methanol to remove traces of moisture and then using

dichloromethane to remove organics. The scintillation vials were combusted for 4 hr at

400 °C and stored in a dessicator prior to use. The analytical balance was calibrated with

standard weights prior to use. A scintillation vial was removed from the dessicator, labeled,

and weighed. Approximately five (5) grams of sample was placed in the vial and the weight

recorded. This procedure was repeated for all samples in the sample set. The samples were

dried for 24 hr in a drying oven set at 63 - 65 °C. The samples were removed from the oven

after the initial 24 hr and allowed to cool to room temperature in a dessicator for at least 30

min. The samples were weighed and the weight recorded. The samples were put back in the

oven for at least 2 hr after which they were removed from the oven and allowed to cool for at

least 30 min in a dessicator. The sample set was reweighed and the weight recorded. If the

difference between the first and the second weighing was less than ± 0.02 g, the dry weight

14
percent was calculated based on the last weighing. The samples were reheated and reweighed

until the difference between weighings was less than ±0.02 g.

4. CALCULATIONS

4.1. Percent dry weight

Percent dry weight = 

[Vial wt. + Dry sample wt.] - [Vial wt.]

[Vial wt. + Wet sample wt.] - [Vial wt.] x 100

4.2. Relative Percent Difference between duplicates

RDP = 

| First value - Second value |

(First value + Second value)/2 x 100

5. QUALITY CONTROL

A method blank and a duplicate were processed for every 20 samples or less. The method

blank was an empty vial. The sample identified for the matrix spike/matrix spike duplicate

sample or the original/duplicate sample for trace organic extraction was used as the percent

dry weight duplicate. For the blank, the absolute difference between the vial weight before and

after the drying was no more than ±0.02 g.

The Relative Percent Difference (RPD) was determined for the calculated percent dry weights

for the original and duplicate. The RPD should agree within ±25%. If the RPD criteria was not

met, the samples were reweighed. If the RPDs were still not within specifications, subsamples

for percent dry weight were redone.

6. CONCLUSIONS

Sediment samples were normally 40 to 70% dry weight. Dry weight measurements were used

to calculate sediment analyte concentrations on a dry weight basis.

15
Determination of Percent Dry Weight for Tissues

Y. Qian and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

The percent dry weight of a tissue is the weight of solid material (in percent) in the

sample compared to the total weight of the sample (dry material plus water). A

procedure used to determine the percent dry weight of biological tissue samples is

described. An aliquot of 0.5 - 1 g of sample was brought to constant weight at 63 - 65

°C. The difference between the weight of the dried sample and that of the wet sample

was used to calculate the percent dry weight of the sample. Quality controls used to

ensure the accuracy and precision in percent dry weight determination are also

described.

1. INTRODUCTION

The percent dry weight of a tissue is the weight of solid material (in percent) in a sample of

biological tissue compared to the total sample weight.

A subsample of tissue was weighed. The subsample was then dried at 63 - 65 °C

(approximately 24 hr) to constant weight. The dry subsample was reweighed.

2. APPARATUS AND MATERIALS

2.1. Apparatus

Balance, top loading with an accuracy of 0.001 g, calibrated daily

Convection oven, 63 - 65 °C

Electrobalance, Cahn or equivalent, with an accuracy of 0.0001 mg, set on the 250 mg - 1

μg scale, calibrated daily

Freeze dryer, LabConco 8 or equivalent

2.2. Labware

Aluminum foil, heavy duty

Beakers, 10 mL capacity, borosilicate glass, heated at 103 - 105 °C for 1 hr and cooled to

room temperature in a dessicator before use.

Cutting tools (scissors, scalpels, etc.), stainless steel, washed, dried and rinsed with

dichloromethane

Forceps, stainless steel, washed, dried and rinsed with dichloromethane

Jars, glass, 1 pint capacity,

Spatulas, stainless steel, washed, dried and rinsed with dichloromethane

Tweezers, stainless steel, anti-magnetic for microbalance

All glassware was washed and solvent rinsed or combusted at 440 °C for 4 hr.

16
2.3. Solvents and reagents

Dichloromethane, Burdick and Jackson pesticide grade or equivalent, lot tested.

3. PROCEDURE

Combusted beakers were prepared for use by heating at 440 °C for 4 hr and then cooling to

room temperature in a dessicator.

It was essential that the dried beakers and dried samples be stored in the dessicator during this

procedure to minimize condensation of moisture which would effect the final weight.

A dried beaker was removed from the dessicator using forceps and was immediately placed on

the top loading balance and weighed. The sample was mixed until it was homogeneous. A

subsample (0.5 - 1 g) of the homogenized tissue was weighed into the beaker. Any unusual

sample characteristics (i.e., odor, oil) was noted in the comment section of the dry weight

bench sheet.

A method blank was prepared by using an empty beaker. The duplicate samples for this method

were the samples designated as matrix spike (MS) and the matrix spike duplicate (MSD)

samples for organic extraction. If MS/MSD samples were not being prepared, then the samples

designated as original/duplicate samples for organic extraction may serve as the quality

control duplicate required for this procedure.

All samples were placed in the a cardboard box lined with aluminum foil using forceps. The box

was loosely covered with aluminum foil and placed in a convection oven at 63 - 65 °C for 24

hr.

The box containing the samples was removed from the oven. The samples were placed in a

dessicator using forceps. The samples were allowed to cool to room temperature in the

dessicator for 30 min.

The dried beakers and dried samples were stored in the dessicator during this procedure to

minimize condensation of moisture which would effect the final weight.

The dried sample in the beaker was removed from the dessicator using forceps and immediately

placed on the top loading balance and weighed. The samples were reheated to 63 - 65 °C for at

least 2 hr, and again cooled in a dessicator for 30 min to room temperature.

The dried sample in the beaker was removed from the dessicator using forceps and immediately

placed on the top loading balance and weighed.

If the difference between the first and second weight of the 10-mL beaker with the dried

sample was less than ±0.02 g, the percent dry weight was calculated according to Section 4.1

using the second weight. If the difference was greater than 0.02 g, the heating, cooling and

weighing process was continued until the difference in the last two weights was less than ±0.02

g, and the percent dry weight was calculated based on the last weighing.

The relative percent difference (RPD) for the percent dry weight between MS and MSD or

Sample and Duplicate was calculated. The acceptable limits for the RPD was within ±25%. If the

RPD was not within specifications, the samples were reweighed. If the RPDs were still not

within specifications, the percent dry weights were redetermined. For the blank, the acceptable

17
limit for the absolute difference between the vial weight before and after drying was less than

±0.02 g.

4. CALCULATIONS

4.1. Percent dry weight

Percent dry weight = 

[Vial wt. + Dry sample wt.] - [Vial wt.]

[Vial wt. + Wet sample wt.] - [Vial wt.] x 100

4.2. Relative percent difference between duplicates

RDP = 

| First value - Second value |

(First value + Second value)/2 x 100

5. QUALITY CONTROL

All quality control samples were processed in a manner identical to actual samples.

A method blank and a duplicate were processed with every sample set. The method blank was an

empty 10-mL beaker. For the blank, the absolute difference between the vial weight before and

after drying should be less than ±0.02 g for the toploading balance. The RPD for the percent dry

weights should agree within ±25%.

6. CONCLUSIONS

Bivalve tissues are normally about 20% dry weight and 80% water. Dry weight measurements

were used to calculate tissue analyte concentrations on a dry weight basis.

18
Sediment Grain Size Analysis - Gravel, Sand, Silt and Clay

S. T. Sweet, S. Laswell, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

Contaminants generally are found in higher concentrations on small-sized particles.

Grain size is an important characteristic of sediments that may be correlated with

contaminant concentrations. This procedure describes the method used to determine

grain size for the NOAA National Status and Trends Program (NS&T).

1. INTRODUCTION

Sediment texture is an important variable in the evaluation of contaminant concentrations and

benthic systems. Numerous studies have shown a correlation between contaminant

concentration and grain size. In benthic ecosystem studies, cross correlations between stations

are often dependent upon substrate characteristics.

In the NOAA NS&T program, attempts are made to collect only fine-grained sediments. Sand

(plus any gravel) is determined by wet sieving with a 62.5 μ screen.

The most common method for the analysis of silt and clay sized particles is the pipette method

(Folk, 1974). It is based on the settling velocity of particles, usually computed on the basis of

Stokes' Law. At specified times, small volumes of a suspension of the silt and clay fraction are

withdrawn, the water evaporated, and the residue weighed.

2. SAMPLE COLLECTION, PRESERVATION AND STORAGE

2.1. Sample collection

Sediment was collected and placed in plastic Ziploc or Whirl-Pak bags, sealed and labeled.

2.2. Sample preservation and storage

Samples were refrigerated. It is recommended that samples not be frozen or freeze-dried as

these processes can cause changes in the grain size distribution.

3. APPARATUS AND MATERIALS

3.1. Labware and apparatus

The following labware and equipment is needed to perform the grain size analyses:

Bags, Ziploc, gallon size. Dow, Indianapolis, IN.

Balance, analytical, 0.1 mg accuracy, Fisher Scientific, Pittsburg, PA.

Beakers, 50-mL

19
Cylinders, graduated, 1-L

Dessicator, Boekel, Philadelphia, PA.

Mason jars, 1 pint, 70610-00518. Kerr Manufacturing Corp., Los Angeles, CA.

Ovens, drying, maintained at 40 - 50 °C and 100 - 130 °C. 1305M. VWR Scientific,

Westchester, PA.

Pipette, 20-mL capacity

Rods, stirring, glass.

Shaker table, H4325, Humboldt Mfg. Co., Norridge, IL.

Sieve, 8-in diameter, 63 mm, ASTME-11 specification. Scientific Products, McGraw Park,

IL.

Sieve, size 10, 2000 mm, -1 phi for gravel, ASTME-11 specification. Scientific Products,

McGraw Park, IL.

Sieve, size 230, 63 mm, +4 phi for sand, ASTME-11 specification. Scientific Products,

McGraw Park, IL.

Timer, 1 sec intervals

Whirl-Paks, 18-oz. NASCO, Ft. Atkinson, WI.

Volumetric glassware and analytical balances were calibrated.

3.2. Reagents

Sodium hexametaphosphate solution, 2.5 g/L. [(NaPO3)6] [10124-56-8] Mallinckrodt,

Paris, KY.

Distilled water

Hydrogen peroxide (30%) (H2O2) [7722-84-1] Mallinckrodt, Paris, KY.

4. PROCEDURE

4.1. Preparation of samples for dry-sieving and pipette analysis

The samples were homogenized by kneading the sample bag by hand. Approximately 15 - 20 g of

sample was placed in a large glass jar. This sample size was chosen to minimize the interaction

of individual grains with each other during settling and lessening the probability of flocculation;

as well as to maximize the amount of material weighed (i.e., with small samples the error in

weighing becomes large with respect to the sample weight). The sample was treated with

approximately 50 - 100 mL of 30% hydrogen peroxide (volume varies with amount of organic

matter present) for 12 hr prior to analyses to oxidize the organic matter present in the

sediment. The sample was washed with distilled water to remove soluble salts. Four hundred

mL of sodium hexametaphosphate solution (approximately 2.5 g/L) was added to disperse the

particles in the sample. The samples were shaken in the shaker table for approximately 24 hr.

4.2. Size analysis of sand/gravel fraction by wet-sieving

A 62.5 μ screen was placed over a 1-L graduated cylinder. The sample containing the dispersed

sediment was poured over the screen and washed with dispersant to rinse any remaining finegrained sediment into the cylinder. This process separated the gravel/sand fraction (on the

screen) from the silt/clay fraction (in the cylinder). The coarse fraction was washed into a

pre-weighed beaker with distilled water and placed in an oven (100 - 130 °C) to dry for 24 hr.

The beaker was removed from the oven and left to cool to room temperature. The beaker was

left open for several hours to allow equilibration with the moisture content of the atmosphere.

The beaker was weighed to 0.1 mg with an analytical balance. This is the gravel/sand fraction.

20
4.3. Silt/clay sized material by settling

The volume of liquid in the graduated cylinder containing the silt/clay material was taken to

exactly one liter volume with dispersant solution. The cylinder was stirred vigorously and left

to stand for one day. When no flocculation was observed, analyses continued. If flocculation was

observed, the sample was discarded and the procedure restarted.

The fine fraction subsamples were taken at the specific times given below to produce the 4 

and 8 φ interval values. Two labeled beakers were pre-weighed to 0.1 mg for use with these

subsamples.

The cylinder was stirred vigorously starting at the bottom and working up until all the sediment

was distributed uniformly throughout the cylinder. At the end of the vigorous stirring, long,

smooth strokes the full length of the cylinder (from the bottom until the stirring rod breaks the

surface) were used. As soon as the rod emerged for the last time, the timer was started. The

pipette was inserted to a depth of 20 cm, and at the end of exactly 20 sec, 20 mL (this is the 4

φ aliquot) was withdrawn. This was the most important single step, since this is the basis for

the determination of total weight of the silt and clay. The suspension was pipetted into a

preweighed beaker. The pipette was rinsed with 20 mL of distilled water and the rinse water

was added to the same beaker.

After exactly 2 hr and 3 min, a 20-mL aliquot at a depth of 10 cm was withdrawn. This aliquot

represents the clay fraction (8 ). The aliquot of the suspension was pipetted into a different

pre-weighed beaker, rinsed with 20 mL of distilled water and added to the beaker. After 2 hr

and 3 min all the silt had settled below 10 cm depth leaving only clay.

The beakers were placed in an oven and the suspensions evaporated to dryness for at least 24

hr at 100 - 130 °C. After 24 hr, the beakers were removed from the oven and left to cool to

room temperature for approximately 4 hr to equilibrate with the moisture content of the

atmosphere. The beakers were weighed to 0.1 mg with an analytical balance, and the weights

recorded on a data sheet.

5. CALCULATIONS

The 4 and 8 φ dry weight aliquots included the weight of the added dispersant. The dispersant

weight (2.5 g/L) was multiplied by the fraction of the total solution removed (20 mL/1000 mL)

to calculate the weight of dispersant (0.05 g) in the 4 and 8 aliquots. The weight of the

dispersant was then subtracted from the 4 and 8 aliquot weight. This total was then

multiplied by 50 (1000 mL/20 mL) to yield the sample weight of the silt plus clay fraction (4

) or the clay fraction (8 ). The sample weight of the silt fraction was calculated by

subtracting the sample weight of the clay fraction (8 ) from the sample weight of the silt plus

clay fraction (4 ).

Total dry weight = wt. sand + wt. silt + wt. clay

where "wt. sand" is the sum of the sand and gravel fractions (particles greater than 62.5 μ).

6. CONCLUSIONS

Results were reported as percent sand, silt, and clay on a dry weight basis. The minimum

method performance standard for the method was detection of 0.5% of each fraction. Duplicate

21
samples were analyzed with every 20 samples. Results were reported to three significant

figures. The grain size analyses is important because there is sometimes a correlation between

sediment contaminant concentrations and percent of fine sediments.

7. REFERENCE

Folk, R. L. (1974) Petrology of Sedimentary Rocks. Hemphill Publishing Company, Austin,

Texas. 182 pp.

22
Total Organic and Carbonate Carbon Content of Sediments

S. T. Sweet and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

This chapter describes the analytical method used to determine total organic and

carbonate carbon in sediments collected as part of the NOAA National Status and Trends

Mussel Watch Project.

1. INTRODUCTION

Total organic and carbonate carbon are parameters that are often useful in providing a better

understanding of sediment contaminant data. The total carbon contained in estuarine sediment is

divided into two fractions: the carbon that originates from plants and animals (organic) and

carbon normally present as calcium carbonate (inorganic).

Total carbon compounds in samples were decomposed by pyrolysis in the presence of oxygen

and the CO2 that was formed was quantified by infrared detection. Total organic carbon (TOC)

was determined after sample acidification which converted carbonate carbon in samples to

carbon dioxide. The CO2 produced was purged from the acidified sample prior to analysis.

Carbonate carbon or total inorganic carbon (TIC) was determined as the difference between

total carbon and total organic carbon.

2. APPARATUS AND MATERIALS

2.1 Equipment

Balance, analytical, capable of weighing to 1 mg, AC 1205. Sartorius, Bohemia, NY.

Crucibles, combustion, 528-018. LECO Corp., St. Joseph, MI.

Detector, infrared, Horiba PIR-2000 or other suitable detector. Horiba, Irvine, CA.

Flow controller, 42300513. Veriflo Corp., Richmond, CA.

Furnace, induction, 523-300. LECO Corporation, St. Joseph, MI.

Integrator, HP-3396A. Hewlett Packard, Avondale, PA.

Mortar and pestle, 500-mL or other suitable container.

Oven, drying, capable of maintaining 40 to 50 °C, 1305M. VWR Scientific, West Chester,

PA.

Pipettes, glass.

Rotameter, 112-02. Cole-Palmer, Inc., Niles, IL.

Scoop, glass measuring, 503-032. LECO Corp., St. Joseph, MI.

Tubes, jet combustion, 550-122. LECO Corp., St. Joseph, MI.

Variac transformer.

23
2.2 Reagents

Accelerator, copper metal, 501-263. LECO Corp., St. Joseph, MI.

Accelerator, iron chip, 501-077. LECO Corp., St. Joseph, MI.

Catalyst Pellets, platinized silica, 501-587. LECO Corp., St. Joseph, MI.

Hydrochloric acid (HCl) [7647-01-0], ACS reagent grade, A144-212. Fisher Scientific,

Pittsburgh, PA.

Magnesium perchlorate (anhydrone) [Mg(CIO4)2] [10034-81-8], 501-171. LECO Corp., St.

Joseph, MI.

Manganese dioxide (MnO2) [1313-13-9], 501-060. LECO Corp., St. Joseph, MI.

Molecular sieve 5A Supelco #2-0301. Belefont, PA.

Standards, pin and ring carbon, range 0.1 to 1.0% carbon, 501-502, 501-503, 501-504.

LECO Corp., St. Joseph, MI.

Water, HPLC grade, 6795-09. Malinckrodt, Paris, KY.

3. Procedure

3.1. LECO system preparation

The LECO induction furnace was allowed to warm up for at least 30 min. The oven was closed,

the oxygen cylinder valve opened, and the regulator set to 40 psi. The oxygen flow was allowed

to stabilize for at least 15 sec before the flow was adjusted to approximately 800 mL/min

using the flow controller. After 30 sec, the panel meter on the Horiba Infrared Analyzer was

set to zero.

3.2. Total carbon determination

3.2.1. Sample preparation

Between 0.1 to 0.5 ± 0.001 g of oven-dried, finely ground homogenized sediment was weighed

on a calibrated balance into a tared, carbon-free combustion crucible. The amount of sample

depended on the expected carbon concentration. Between 0.5 and 8.6 mg of carbon were

required for a response within the range of the standard curve.

One scoop (approximately 1.4 g) each of the copper and iron chip accelerators were added to

each of the crucibles containing samples. All crucibles were kept covered with aluminum foil

prior to analyses.

3.2.2. Sample analyses

The crucible was placed on the oven pedestal and sealed within the oven combustion tube. The

oxygen flow was allowed to stabilize for about 15 sec, the flow rate checked on the rotameter,

and adjusted to 800 mL/min. The induction furnace was turned on. The carbon present in the

sample began to combust after about 20 sec and the evolved CO2 was trapped on a 5A 45/60

mesh molecular sieve. The Variac transformer was set at 40% and switched on. After the

sieve had heated for one minute, the Hewlett Packard integrator was started. Once the signal

returned to baseline, the Variac was shut off, the cooling fan turned on, and the integrator

stopped. The peak area for the sample was recorded. The oven was opened and the hot crucible

was removed. This procedure was repeated for all samples in each analytical batch.

24
3.2.3. Standard analyses

Standard LECO pin and ring carbon standards were placed into an empty carbon free combustion

crucibles and one scoop of the copper accelerator was added. LECO calibration standards

consisted of 1 g steel rings or pins of precisely known carbon concentration. These calibration

standards were traceable to NIST standard reference materials. Different LECO carbon

standards were chosen to cover the range of 0.1 to 1.0% carbon and at least five different

carbon standards were analyzed with each sample set. Standards were analyzed using the same

procedure as outlined in Section 3.2.2.

3.3. Total Organic Carbon determination

3.3.1. Sample preparation

Appropriate amounts of dried sample (Section 3.2.1) were weighed into a tared crucible. The

samples were acidified by adding 10% HCl in a dropwise fashion until bubbling stopped. The

acidified samples were dried overnight at 50 °C in a drying oven.

3.3.2. Sample analyses

These samples were analyzed as described in Section 3.2.2.

3.4. Total carbonate carbon content

Carbonate carbon was determined by difference between total carbon and total organic carbon

in a sample.

4. STANDARDIZATION AND CALCULATIONS

Prior to analyzing samples, standards were analyzed (daily) to establish a standard curve.

Standard curves varied slightly from day to day.

A set of five different LECO carbon standards containing a known range of carbon were

analyzed to establish the curve. Several standard rings and/or pins were run initially to bring

the system to correct operating conditions. The calibration curve was prepared by plotting

percent carbon versus standard peak areas.

The best fit equation for the calibration curve was determined by regression analysis. If the

correlation coefficient for the equation was less than 0.99, the standards data set was

discarded and another set of five calibration points analyzed. The calibration curve was used to

determine the carbon content of samples analyzed that day.

The sample peak areas obtained from the integrator were converted to percent carbon using a

second order equation obtained from the calibration curve

Cs = 

M1A + M 2A2 + b

wt.

where A was the area of the sample peak, b was the intercept of the second order calibration

equation, M1 and M2 were the coefficient from the second order calibration equation, Cs was

the percent carbon in the sample, and wt was the sample dry weight.

Percent carbonate carbon = Percent total carbon - Percent TOC

25
5. QUALITY CONTROL

Quality control samples were processed and analyzed in an identical manner to that used for the

samples.

A method blank, consisting of approximately 1.4 g each of copper and iron chip accelerators,

was run with every 20 samples or every sample set, whichever was more frequent. Blank

levels were less than three times method detection limit (MDL).

Duplicate samples were run for every 20 samples or at least with every sample set. Duplicates

agreed within ±20% for low level (<1.0% carbon) samples and ±10% for normal/high level

( 1.0% carbon) samples. Duplicates were less precise for very inhomogeneous samples (i.e.,

peats, samples containing twigs, grasses, etc.).

LECO pin and ring carbon standards were used as reference materials and standards.

6. REPORTING AND PERFORMANCE CRITERIA

Reporting units were percent organic carbon on a dry weight basis and percent carbonate

carbon on a dry weight basis. Results were reported to two significant figures.

The minimum method performance standard was detection of 0.02% carbon in a sample.

7. CONCLUSIONS

Total organic and carbonate carbon were determined in sediments collected along the U.S. coast

for use in the interpretation of organic contaminant concentrations of samples collected as part

of the NOAA National Status and Trends Mussel Watch Project.

26
Determination of Percent Lipid in Tissue

Y. Qian, J. L. Sericano, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

The percent lipid (weight/weight basis) of a tissue is operationally defined as the

weight (in percent) of the material extracted by dichloromethane from the tissue

sample. The procedure used to determine the percent lipids of biological tissue samples

is described. An appropriate amount of sodium sulfate-dried tissue sample was

extracted three times with dichloromethane (100 mL each time). An aliquot of 20 mL of

the extract was quantitatively removed for lipid determination. This aliquot was

further dried with sodium sulfate and brought to a final volume of 1.0 mL in

dichloromethane. An aliquot of 100 μL was taken and evaporated to constant weight.

The residual weight of this dried 100 μL portion was used to calculate the percent lipids

of the sample based on the dry weight. Quality control measures used to ensure the

accuracy and precision of percent lipids determination are also described.

1. INTRODUCTION

The percent lipid content of a tissue is operationally defined as the weight (in percent) of

material extracted from a tissue with dichloromethane.

The tissue sample was extracted with dichloromethane in the presence of anhydrous sodium

sulfate. An aliquot of the extract was removed for lipid determination. This aliquot was filtered

and concentrated to a known volume. A known volume of the aliquot was taken, evaporated to

dryness, weighed, and the percent lipids for the sample was determined on a dry weight basis.

2. APPARATUS AND MATERIALS

2.1. Apparatus

Electrobalance, Cahn Electrobalance or equivalent, with an accuracy of 0.0001 mg, set on

the 250 mg - 1 μg scale

Hot plate, set on low heat

Micropipette, 100 μL

Rotoevaporator, Brinkmann Rotovapor R110, or equivalent

Vortex mixer

2.2. Labware

Aluminum foil, heavy duty

Flat bottom flasks, 125-mL capacity, borosilicate glass

Funnels, powder, 65 cm, borosilicate glass

Glass fiber filters, 11.0 cm, Whatman GF/C or equivalent

Graduated cylinders, 25-mL and 500-mL capacity, borosilicate glass

Syringe, 1-mL volumetric

Tweezers, stainless steel, anti-magnetic

Vials, 7 dram capacity, borosilicate glass, with Teflon-lined, solvent rinsed caps

27
All glassware was washed and then solvent rinsed or combusted at 440 °C for 4 hr.

2.3. Solvents and reagents

Dichloromethane (CH2Cl2) [75-09-2], Burdick and Jackson pesticide grade or equivalent,

lot tested.

Sodium sulfate (Na2SO4) [7757-82-6], granular, anhydrous, J. T. Baker, ACS reagent

grade or equivalent, combusted at 440 °C for 4 hr; stored at 120 °C and cooled to

room temperature in a dessicator before use.

3. PROCEDURE

After tissue samples were extracted (see tissue extraction method), the solvent level of the

sample extract was marked on the 500-mL flat-bottom flask. The tissue extract was mixed by

swirling the 500-mL flat-bottom flask and a 20-mL aliquot of the tissue extract was removed

using a 25-mL graduated cylinder for lipid determination. A glass fiber filter containing

approximately 10 g of anhydrous sodium sulfate was pre-wetted with dichloromethane and the

20-mL aliquot was filtered into the 125-mL flask. The graduated cylinder was rinsed three

times with 3 - 5 mL of dichloromethane and the rinses poured through the filter. The total

volume of the sample extract was determined after the sample extract was evaporated and

transferred from the 500-mL flat-bottom flask. Each of the 500-mL flasks was filled with tap

water to the previously marked line, and the volume of the water measured with a 500-mL

graduated cylinder.

A method blank and a duplicate were processed with every sample set. Twenty (20) mL of the

extraction method blank was used as the lipid method blank. The sample extracts for the

extraction batch matrix spike (MS)/matrix spike duplicate (MSD) was used as the

original/duplicate.

The filtered aliquot was evaporated using the Rotoevaporator to near dryness. The residue was

quantitatively transferred with dichloromethane to a 7-dram vial. The solvent in the 7 dram

vial was evaporated to dryness under a stream of nitrogen. Alternatively, the solvent was

allowed to evaporate by loosely capping the vials.

A 1-mL syringe was rinsed three times with dichloromethane. Dichloromethane was added to

the lipid vial so that the final volume was 1 mL. The cap was replaced and the sample vial was

shaken by a vortex mixer for 10 sec. The vial was allowed to sit for at least 30 min and was

mixed using the vortex mixer again. For samples that were very high in lipid, it was necessary

to adjust the final volume of 2 mL.

The 100 μL micropipette was calibrated according to the manufacturer's instructions. The

micropipette was pre-rinsed at least five times with dichloromethane. The electrobalance was

calibrated with the standard 200 mg weight according to the manufacturer's instructions.

Using tweezers, a small piece (approximately 1 cm x 2 cm) of glass fiber filter was placed on a

corrugated aluminum foil support on the hot plate for a few minutes. The filter was transferred

to the electrobalance weighing pan and the balance tared. The filter was placed back on the hot

plate.

A 100-μL aliquot of the lipid extract was removed using a 100 μL and slowly dotted onto the

warm filter. The micropipette was rinsed again at least five times with dichloromethane. The

7-dram vial was tightly capped and stored in the freezer at -20 °C.

28
When the solvent had evaporated from the filter, the filter was placed on the weighing pan of

the tared electrobalance using tweezers. When the reading was stable, the weight (in mg) was

recorded.

The percent lipids in the extract was calculated on a dry weight basis.

The relative percent difference (RPD) for the percent lipid values for the original and duplicate

sample were calculated. The acceptable RPDs for the sample and Duplicate or MS and MSD was

required to be within ±25%. If the RPD was not within ±25%, the original and duplicate sample

extracts were reweighed. If the RPDs were still not within specifications, the samples were

re-extracted for lipids. The lipid weight (in mg) for the blank was less than 0.005 mg.

4. CALCULATIONS

4.1. Percent lipid

Percent lipid = 

TV

AV x 

FV

VW x 

LW

SW x 100

where TV is the total volume of the extract (mL), AV is the volume of the aliquot (mL), FV is

the final volume (mL), VW is the volume weighed (mL), LW is the lipid weight (grams), and SW

is the sample weight (grams).

4.2. Relative percent difference (RPD) for duplicate analysis

RDP = 

| First value - Second value |

(First value + Second value)/2 x 100

5. QUALITY CONTROL

All quality control samples were processed in a manner identical to the actual samples.

A method blank and a duplicate were processed with every sample set. The lipid method blank

was a 20-mL aliquot of the extraction method blank. The extraction batch sample extract

designated as the MS/MSD or as original/duplicate was used as the QC duplicate required for

this procedure. The RPD for the calculated percent lipid values for the duplicates was

determined. The RPD agreed within ±25%. The lipid weight (in mg) for the method blank was

less than 0.005 mg.

6. CONCLUSIONS

This method operationally defined the percent lipid in tissue by extraction with

dichloromethane. Lipid content has been found to be correlated to contaminant concentrations

for specific tissues and whole organisms.

29
30
TRACE ELEMENT ANALYSES

31
TERL Trace Element Quantification Techniques

B. J. Taylor and B. J. Presley

Oceanography Department

Texas A&M University

College Station, TX

ABSTRACT

Sample preparation and analysis methods have been developed and refined that allow

the accurate and precise determination of major and trace elements in sediment and

biological tissue samples. Sample preparation emphasizes homogenization and total

digestion steps that minimize contamination. Analysis utilizes atomic absorption and

neutron activation techniques, and includes a full suite of quality assurance samples

(with an emphasis on certified reference materials) in order to produce reliable data.

These methods allow measurement of both background and elevated concentrations

within NOAA’s Status and Trends Program, permitting subtle temporal and spatial

differences to be detected.

1. INTRODUCTION

This chapter documents the analytical procedures used for major and minor element analysis of

marine sediments and tissue samples collected from the Gulf of Mexico coast of the United

States as part of the Mussel Watch Project of NOAA's National Status and Trends Program.

These procedures were used by the Trace Element Research Laboratory (TERL), Department of

Oceanography, Texas A&M University to analyze samples collected from 1991 to 1997.

2. EQUIPMENT AND SUPPLIES

2.1. Instrumentation

Perkin-Elmer model Z/3030 atomic absorption spectrophotometer. Perkin-Elmer, Norwalk, CT.

Printer, Okidata Microline 184

Graphite furnace, Perkin-Elmer HGA-600

Graphite furnace cooling unit:

Constant Temperature Circulator Model FK. Haake, Paramus, NJ.

IC-6 refrigeration unit, Lauda water bath. Curtin-Matheson, Houston, TX.

Autosampler, Perkin-Elmer AS-60

EDL power supply, Perkin-Elmer Model 040-0354

Perkin-Elmer model 4100-ZL atomic absorption spectrophotometer. Perkin-Elmer, Norwalk, CT

Printer, Okidata Microline 320

Graphite furnace, transversely-heated stabilized temperature platform

Autosampler, Perkin-Elmer AS-70

EDL power supply, Perkin-Elmer System 2

Perkin-Elmer model 3110 atomic absorption spectrophotometer. Perkin-Elmer, Norwalk, CT

Background correction, deuterium arc

Burner (0040-0146) with standard nebulizer, flow spoiler, and single-slot acetylene

(0040-0266) and nitrous oxide (0040-0277) burner heads

Digital absorbance readout

32
Mercury Monitor, Model 1235. Laboratory Data Control Analytical, Riviera Beach, FL.

Wavelength setting, 254 nm

Absorbance cell, 30 cm

Recorder, OmniScribe Model A5101-2. Houston Instruments, Houston, TX.

Ortec pure germanium large volume co-axial detectors, GEM-22170S and 23185-P, with 1.68

KeV and 1332.5 KeV resolution, 22% efficient compared to NaI detector. EG&G Ortec, Oak

Ridge, TN.

Nuclear Data model 9900 MCA, implemented on a VAX station II-GPX.

2.2. Supplies

Hollow cathode lamps (HCL). Perkin-Elmer, Norwalk, CT.

Electrodeless discharge lamps (EDL). Perkin-Elmer, Norwalk, CT.

Argon, high-purity (99.999%)

Acetylene, industrial

Nitrous oxide

Graphite tubes, pyrolytically coated, grooved, Perkin-Elmer B010-9322

Graphite tubes, pyrolytically coated, ungrooved, Perkin-Elmer B010-9322

L'vov graphite platforms, pyrolytically coated, Perkin-Elmer B010-9324

Autosampler cups, 2-mL, polystyrene, B2713-2. Baxter Scientific Products, McGaw

Park, IL.

2.3. Labware

Balance, 0.01 g, Fisher 200 Ainsworth

toploader. Fisher Scientific, Pittsburgh,

PA.

Jars, large, Teflon

Pipette tips, for Finnpette, Finntip 62.

Labsystems

Balance, 0.01 g, Mettler PC2000. Pipette tips, polypropylene for Eppendorf

pipettes, 10 - 100-mL Model 22 34190-1

and 200-1000 mL, 22 35 090-1.

Balance, analytical, 0.0001 g, Mettler H10.

Balls, 1-cm diam., Teflon

Balls, 3.5-cm diam, Teflon Pipette, Finnpette, adjustable, 1000 - 5000-

mL 9402020. Curtin-Matheson Scientific,

Houston, TX.

Bench, clean, with HEPA filter. Liberty

Industries, East Berlin, CT.

Bottles, screw-cap bottles, polyethylene,

wide-mouth, 1-oz., Nalgene 2104-0001

Pipette, transfer, polyethylene.

Pipettes, Eppendorf, fixed volume: 10-mL,

22350102; 25-mL, 22350307; 50-mL,

22350404; 100-mL, 22350501; 200-

mL, 22350609; 500-mL, 22350706;

1000-mL, 22350803.

Digestion vessels, 50-mL, Teflon (PFA), 561-

R. Savillex, Minnetonka, MN.

Drying oven, 60 °C, NAPCO 332. CurtinMatheson Scientific, Houston, TX.

Drying oven, 130 °C, Thelco Vials, snap-cap vials, polystyrene, 5-, 15-,

and 40-dram. Baxter Scientific Products,

McGaw Park, IL.

Freeze dryer (Virtis 10-100) and vacuum

pump (Welch Duo-Seal 1402B80). Virtis

Co., Gardner, NY, and Welch, Skokie, IL.

2.4. Reagents

Ammonium dihydrogen phosphate (NH4H2PO4)

[7722-76-1], Spectropure Grade, P30.

Spex, Edison, NJ.

Ascorbic acid (C6H8O6) [50-81-7], A-7506.

Sigma, St. Louis, MO.

33
Boric acid (H3

BO3) [10043-35-3], 10659,

Grade 1. Johnson Matthey, West Chester,

PA.

Nitric acid (HNO3) [7697-37-2], concentrated

(70%), 2704-7x6. Mallinckrodt, Paris,

KY.

Citric acid (C6H8O7) [77-92-9], 0110. J. T.

Baker, Phillipsburg, NJ.

Nitric acid (HNO3) [7697-37-2], concentrated

(70%), Ultrex 6901-05. J. T. Baker,

Hydrochloric acid (HCl) [7647-01-0], Phillipsburg, NJ.

concentrated (37%), Ultrex 6900-05. J.

T. Baker, Phillipsburg, NJ.

Palladium metal [7440-05-3], Specpure,

560001. Johnson Matthey, West Chester,

Hydrofluoric acid (HF) [7664-39-3], PA.

concentrated (48%), 9560-06. J. T.

Baker, Phillipsburg, NJ.

Perchloric acid (HClO4) [7601-90-3], concentrated (70%), Ultrex 4805-01. J. T.

Magnesium nitrate [Mg(NO3)2 Baker, Phillipsburg, NJ. .6H20] [13446-

18-9], MG60-50. Spex, Edison, NJ. Stannous chloride, (SnCl2

.

 2H2O) [10025-69-

Nickel oxide (Ni0) [1313-99-1], powder. 1], 8176. Mallinckrodt, Paris, KY.

Spex, Edison, NJ. Water, redistilled in quartz sub-boiling still.

2.5. Matrix modifiers

Ammonium phosphate: 0.04 g/mL in quartz-distilled water

Ascorbic acid: 2% w/v made with quartz-distilled water

Citric acid: 2% w/v made with quartz-distilled water

Hydroxylamine hydrochloride

Magnesium nitrate: 0.02 g/mL in quartz-distilled water

Palladium nitrate: 1000 mg Pd/mL made by dissolving 0.05 g Pd metal in 2 mL

Concentrated Ultrex HNO3 and diluted to 50 mL with quartz-distilled water

2.6. Standards

Baxter Ricca standards, 1000 ppm. Ricca Chemical Co., Arlington, TX.

Element Stock number

Ag 7100-16UK

Al 600-16UK

As 800-16UK

Cd 1700-16UK

Cr 2100-16UK

Cu 2300-16UK

Fe 4200-16UK

Hg 4800-16UK

Mn 4600-16UK

Ni 5300-16UK

Pb 4300-16UK

Sb 700-16UK

Se 6700-16UK

Sn 8500-16UK

Zn 9500-16UK

34
3. SAMPLE TREATMENT

3.1. Oyster and mussel tissue

3.1.1. Bivalve shucking

Oysters and mussels were rinsed with distilled water to remove extraneous material and

shucked with a stainless steel knife (using care not to touch the tissue). Tissue was removed

with plastic forceps and rinsed with distilled, deionized water to remove sediment particles

from gills and exterior tissue surfaces. Soft parts were transferred to a tared Ziploc

polyethylene bag, and the number of individuals shucked and placed in the bag is recorded. When

all individuals from a site had been shucked, they were weighed on a top loading balance to

measure the total sample wet weight. The pooled samples were placed in a freezer to await

further processing.

3.1.2. Bulk homogenizing

Ziploc bags containing pooled tissue were removed from the freezer and allowed to thaw. The

entire pooled sample was transferred to an acid-washed Teflon jar and 3 large Teflon balls

were added. The Teflon lids were securely tightened and the jars placed in Ziploc bags and

shaken in an industrial paint shaker for 20 min. After the bulk sample was homogenized, an

aliquot of the sample was transferred to a clean 40 dram snap vial and frozen.

3.1.3. Freeze drying

The frozen aliquot from the bulk homogenization step was placed in a freeze drier and allowed

to dry for several days, depending upon the total mass of tissue being dried at one time. In

some cases it was necessary to remove the samples from the freeze drier and drain

accumulated water from the trap before continuing with the drying step.

3.1.4. Homogenization of dry aliquot

When samples have been thoroughly dried, three small Teflon balls were inserted into each snap

cap vial, the lids were affixed, and the samples placed in a Spex shaker mill for 1 min. The

Teflon balls were then removed, and the samples stored in closed vials until weighing.

3.1.5. Digestion

Approximately 0.2-g samples of dried tissue were weighed to the nearest 0.0001 g and

transferred to tared, acid-washed Teflon bombs. Three mL of HNO3 were added and the bombs

capped loosely and allowed to stand overnight at room temperature. The bombs were then

tightened to 18 foot-lbs and placed in a 130 °C oven for a total of approximately 20 hr. During

this time, the bombs were periodically removed from the oven, allowed to cool, and vented to

release excess pressure. When digestion was complete, the samples were allowed to cool and

18 mL of quartz distilled water added to each sample. The bombs were closed, mixed by

shaking, and weighed to 0.01 g to determine the total solution weight. The digest solution was

transferred to labeled 1-oz polyethylene bottles. Solution density was determined by weighing

known volumes with calibrated Eppendorf pipettes in order to determine solution volume.

For analysis of Hg, tissue samples were digested using a modified version of the Environmental

Protection Agency (EPA) method 245.6. Approximately 0.15 to 0.3 g (dry weight) sample was

weighed into a 50-mL polypropylene centrifuge tube. Concentrated H2SO4 (2.5 mL) and 1.5 mL

of concentrated HNO3 were added and the samples heated in a water bath at 90 - 95 °C for 30

35
min. After cooling, 10 mL of distilled water, 10 mL of 5% (w/w) KMnO4

, and 5 mL of 5%

(w/w) of K2S2O8 were added to each tube, and the samples left overnight without heating.

Before analysis, 5 mL of 10% (w/w) NH2OH . HCl were added to reduce excess permanganate

and the volume brought to 40 mL with distilled water.

3.1.6. Displacement volume

Bivalve shells were removed from the refrigerator and placed in a displacement cylinder

containing distilled water. Water escaping from the cylinder as shells were added was captured

in a graduated cylinder. When the water flow ceased the volume of displaced water was

recorded. Shells were then removed from the cylinder, placed in plastic bags, and returned to

the refrigerator.

3.2. Bottom sediment

Bottom sediment samples were prepared for atomic absorption analysis and activation analysis

by freeze drying and wet digestion.

3.2.1. Homogenization

Wet bulk sediment was stored frozen until sample processing begins. Sediment was thawed and

homogenized with a clean plastic spatula. A homogeneous aliquot of the bulk sample was

transferred to a labeled 40 dram snap cap vial and frozen. The remainder of the sample was

archived in the freezer.

3.2.2. Freeze drying

The snap cap vial containing the sediment sub-sample was placed in a freeze drier for the

period of time required for complete drying. Depending upon the amount of water in the freeze

drier, this ranged from 12 - 76 hr.

3.2.3. Homogenization of dry aliquot

In some cases, homogenization of freeze dried sediment was accomplished by simply placing the

snap cap vials in a Spex shaker. When this is not sufficient, the samples were individually

ground in alumina mortar and pestles and the powdered samples returned to the vials in which

they were freeze dried.

3.2.4. Digestion

Approximately 0.2 g of homogenized, dried sediment were weighed to the nearest 0.0001 g and

transferred to tared, acid-washed Teflon bombs. Three mL HNO3 were added and the bombs

tightened to 18 foot-lbs and placed in a 130 °C oven for a total of approximately 12 hr. During

this time, the bombs were periodically removed from the oven, allowed to cool, and vented to

release excess pressure. After this period, the bombs were removed from the oven and

allowed to cool. Two mL of concentrated HF were added and the bombs retightened and returned

to the oven for 12 hr. After cooling, 18 mL of 4% boric acid were added and the bombs

retightened and returned to the oven for another 12 hr. After the samples were allowed to

cool, the content of the bombs were mixed by shaking, and the bombs weighed to 0.01 g to

determine the total solution weight. The digest solution was then transferred to labeled 1 oz

polyethylene bottles. Solution density was determined by weighing known volumes with

calibrated Eppendorf pipettes in order to determine solution volume. At this point, a 20-fold

dilution was made for FAAS analysis of Al, Fe, Mn, Si, and Zn. One mL of digest solution was

36
diluted with 19 mL of an acidified seawater solution containing 2:1:17 proportions of

seawater:HNO3:deionized water.

For analysis of Hg, sediment samples were digested using a modified version of EPA method

245.5. Approximately 0.1 to 1.0 g (dry weight) sample was weighed into a 50-mL

polypropylene centrifuge tube. Concentrated H2SO4 (2.5 mL) and 1.5 mL of concentrated HNO3

were added and the samples heated in a water bath at 90 - 95 °C for 30 min. After cooling, 10

mL of distilled water, 10 mL of 5% (w/w) KMnO4, and 5 mL of 5% (w/w) of K2S2O8 were

added to each tube, and the samples again heated in a water bath at 90 - 95 °C for 30 min.

Before analysis, 5 mL of 10% (w/w) NH2OH . HCl were added to reduce excess permanganate

and the volume brought to 40 mL with distilled water.

4. CALIBRATION AND ANALYSIS

Calibration standards were prepared by serial dilution of commercially available atomic

absorption standards using calibrated micropipettes, new snap-cap vials, a top loading balance,

and 2 N HNO3. Concentrations of working standards were verified by comparison with

independent standards traceable to National Institute of Standards and Technology (NIST)

Standard Reference Materials.

In all cases, final working standards were prepared in an acid matrix that matches that of the

samples being analyzed. For some elements, it was necessary to further attempt to match the

major ion composition of the samples. This was most apparent in graphite furnace AAS when

the peak shape of the samples was significantly different from that of the standards. For

example, the standards may have a relatively broad, Gaussian-shaped peak while the sediment

samples may have an extremely sharp peak indicative of rapid volatilization of the metal. In

this case, the standards were prepared in a solution that had Si, Al, Fe, Ca, and Mg added at

final concentrations of 3000, 400, 200, 100, and 100 ppb, respectively.

For graphite furnace atomic absorption analysis, standards were placed in positions 1 - 4 of the

autosampler tray, and samples and associated quality control samples in positions 5 - 40.

Matrix modifiers were placed in positions 0 and 40 if necessary. Analysis begins with position

1, thus standards were analyzed first. After the samples in positions 16, 28, and 40, the

standards were rerun before sample analysis continues. After one tray is finished, another

tray was placed on the autosampler and analysis was begun in position 5.

5. CALCULATIONS

Trace metal concentrations were calculated by comparing analytical signals of unknowns with

those of calibration standards, and then multiplying the observed concentration by the

instrumental and digestion dilution factors.

The least-squares fit of the data was calculated, treating Abs (or Abs-sec) as the dependent

variable ("y"), and concentration as the dependent variable ("x"). If the concentration range

extends into the non-linear range, a second order fit was used. The intercept, the first and

second order coefficients (if appropriate), and R, the correlation coefficient, were calculated.

Abs = a + b (concobs)

37
concobs = 

Abs - a

b

5.1. Concentration

The Perkin-Elmer Z3030 used in much of this project incorporates a patented curve-fitting

routine described here. In this method, the concentration was determined as

C = 

K1 A + K 3 A2

K2 A - 1

where C is the concentration, A is the absorbance-sec, and K1, K2, and K3 are the coefficients

determined by solution of simultaneous equations or by the method of least squares.

In our laboratory, the instrument was allowed to auto-select the appropriate equation to fit the

data. In all cases, the standard curve was computed from a blank and three standards that were

equally spaced from zero to the maximum concentration. For example, concentrations might be

0, 1, 2, and 3 ppb. If the highest standard was within 15% of the value expected from

extrapolation of the lowest standard, a 2-coefficient equation was used. If the highest standard

was not within 15% of the value expected from extrapolation of the lowest standard, a 3-

coefficient equation was used. Because of the number of standards analyzed, the 2-coefficient

equation was calculated via least squares regression and the 3-coefficient equation via solution

of simultaneous equations. The instrument had the capability to perform a "reslope" based on a

single point. However, our recalibrations always involved complete re-analysis all standards.

Comparison of observed values predicted by the Perkin-Elmer curve fitting routine with those

calculated independently by least squares showed insignificant differences that were within

rounding errors of the printout.

5.2. Dilution factor

The dilution factor, DF, resulting from sample digestion was calculated using the equation

DF = 

[(bomb tot.) - (bomb tare)]

(spl. wt.) x (soln. dens.)

where bomb tare was the tare weight of the digestion vessel (g); bomb tot. was the total

weight of the digestion vessel plus digest solution (g); spl. wt. was the weight of the dry

sample (g); and soln. dens. was the density of the digest solution (g/cm3).

5.3. Concentration

The concentration in the original sample was calculated according to the relationship:

If concobs < DL, final concentration (DL) (DFinstr) (DFdign)

If concobs DL, final concentration = (concobs) (DFinstr) (DFdign)

where concobs was the concentration observed in the aqueous sample; DL was the detection

limit of the analytical technique; DFinstr was the dilution factor of the analytical technique, if

necessary; and DFdign was the dilution factor of the sample digestion.

38
6. CONCLUSIONS

Through a consistent effort to eliminate sample contamination, increase accuracy, and improve

precision, sample preparation and analysis methods evolved to a level where reliable data can

be generated even at low background levels. Critical points within the overall process include:

• a scrupulous effort to minimize contamination from laboratory dust;

• minimal reuse of materials such as bottles, sample cups, and pipette tips;

• utilization of ultrapure reagents;

• closed digestion systems;

• meticulous attention to detail in both sample preparation and analytical stages; and

• personnel with sufficient experience to make necessary method adjustments.

Generating accurate and precise data on environmental samples requires continuous scrutiny of

instrumental operation and data quality, and is not consistent with a “production”, “just run

the method” mentality. Unless sample concentrations were “known” beforehand, such as from

previous years’ data, the best measures of data quality were certified reference materials and

performance in blind intercalibration exercises.

7. INSTRUMENTAL ANALYSIS

Table 1 provides information for which analytical method was used for each sample matrix.

Table 1. Elemental quantification techniques by matrix.

Matrix Matrix

Section Analyte Method Tissue Sediment Section Analyte Method Tissue Sediment

7.1 Mercury CVAA X X 7.14 Selenium GFAA X X

7.2 Aluminum FAA X 7.15 Tin GFAA X X

7.3 Copper FAA X 7.16 Aluminum INAA X

7.4 Iron FAA X X 7.17 Chromium INAA X

7.5 Manganese FAA X 7.18 Iron INAA X

7.6 Zinc FAA X X 7.19 Manganese INAA X

7.7 Silver GFAA X X 7.20 Arsenic INAA X

7.8 Arsenic GFAA X X 7.21 Chromium INAA X

7.9 Cadmium GFAA X X 7.22 Iron INAA X

7.10 Chromium GFAA X X 7.23 Selenium INAA X

7.11 Copper GFAA X X 7.24 Silver INAA X

7.12 Nickel GFAA X X 7.25 Zinc INAA X

7.13 Lead GFAA X X

CVAA - Cold vapor atomic absorption

FAA - Flame atomic absorption

GFAA - Graphite furnace atomic absorption

INAA - Instrumental neutron activation analysis

39
7.1. Mercury

METHOD: Cold vapor atomic absorption

INSTRUMENTATION: Laboratory Data Control Model 1205 Spectrophotometer with 30-

cm path length gas cell.

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 254 nm

Lamp: Low pressure, hot cathode Hg lamp

Peak measurement: Peak height (absorbance)

Range: 0.2 absorbance units

Output: To strip chart recorder, 10 mV full scale

Reaction Conditions:

Sample volume: 1 mL

Reductant: 10% SnCl2

Reductant volume 0.1 mL

Reaction vessel 25-mL Erlenmeyer flask

STANDARDS: 0, 0.5, 1.0, and 2.0 ppb prepared from 1000 ppm Ricca standard in

0.2 M HNO3 / 0.1 M HCl.

APPROXIMATE SENSITIVITY: Approximately 0.160 Abs for 1 mL of 2 ng/mL solution.

40
7.2. Aluminum

METHOD: Flame atomic absorption

INSTRUMENTATION: Atomic absorption spectrophotometer Perkin-Elmer 3110

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 309.3 nm

Slit width: 0.7 nm

Lamp: Al HCL, 10 mA

Background correction: Deuterium arc

Peak measurement: Peak height (absorbance)

Read mode: Peak

FLAME SETTINGS:

Fuel: Acetylene

Oxidant: Nitrous oxide

Flame: Oxidizing

Burner: Single slot, 5.5 cm, parallel

STANDARDS: 0, 10, 30, and 50 ppm prepared from 1000 ppm Ricca standard in

0.2 M HNO3.

MATRIX MODIFIERS: Samples and standards are spiked with La (prepared from LaCl3) to

a final concentration of 1000-2000 ppm La to suppress ionization

interferences.

APPROXIMATE SENSITIVITY: 50 μg/mL gives approximately 0.200 Abs.

41
7.3. Copper

METHOD: Flame atomic absorption

INSTRUMENTATION: Atomic absorption spectrophotometer Perkin-Elmer 3110

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 324.7 nm

Slit width: 0.7 nm

Lamp: Cu HCL, 15 mA

Background correction: deuterium arc

Peak measurement: Peak height (absorbance)

Read mode: Peak

FLAME SETTINGS:

Fuel: Acetylene

Oxidant: Air

Flame: Oxidizing

Burner: Single slot, 10 cm, parallel

STANDARDS: 0, 1, 2.5, and 5 ppm prepared from 1000 ppm Ricca

standard in 0.2 M HNO3.

APPROXIMATE SENSITIVITY: 2 μg/mL gives approximately 0.125 Abs.

42
7.4. Iron

METHOD: Flame atomic absorption

INSTRUMENTATION: Atomic absorption spectrophotometer Perkin-Elmer 3110

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 248.3 nm

Slit width: 0.2 nm

Lamp: Fe HCL, 18 mA

Background correction: Deuterium arc

Peak measurement: Peak height (absorbance)

Read mode: Peak

FLAME SETTINGS:

Fuel: Acetylene

Oxidant: Air

Flame: Oxidizing

Burner: Single slot, 10 cm, parallel

STANDARDS: 0, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ppm prepared from 1000 ppm

Ricca standard in 0.2 M HNO3.

APPROXIMATE SENSITIVITY: 3 μg/mL gives approximately 0.100 Abs.

43
7.5. Manganese

METHOD: Flame atomic absorption

INSTRUMENTATION: Atomic absorption spectrophotometer Perkin-Elmer 3110

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 279.5 nm

Slit width: 0.2 nm

Lamp: Mn HCL, 20 mA

Background correction: Deuterium arc

Peak measurement: Peak height (absorbance)

Read mode: Peak

FLAME SETTINGS:

Fuel: Acetylene

Oxidant: Air

Flame: Oxidizing

Burner: Single slot, 10 cm, parallel

STANDARDS: 0, 0.4, 0.8, and 1.2 ppm prepared from 1000 ppm Ricca standard

in 0.2 M HNO3.

APPROXIMATE SENSITIVITY: 1 μg/mL gives approximately 0.100 Abs.

44
7.6. Zinc

METHOD: Flame atomic absorption

INSTRUMENTATION: Atomic absorption spectrophotometer Perkin-Elmer 3110

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 213.9 nm

Slit width: 0.7 nm

Lamp: Zn HCL, 15 mA

Background correction: Deuterium arc

Peak measurement: Peak height (absorbance)

Read mode: Peak

FLAME SETTINGS:

Fuel: Acetylene

Oxidant: Air

Flame: Oxidizing

Burner: Single slot, 10 cm, parallel

STANDARDS: 0, 0.5, 1.0, and 2.0 ppm prepared from 1000 ppm Ricca standard

in 0.2 M HNO3.

APPROXIMATE SENSITIVITY: 1 μg/mL gives approximately 0.190 Abs.

45
7.7. Silver

METHOD: Graphite furnace atomic absorption

INSTRUMENTATION:

Spectrophotometer: Perkin-Elmer Z3030

Graphite furnace: Perkin-Elmer HGA 600

Autosampler: Perkin-Elmer AS 60

Printer: Okidata Microline 184

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 328.1 nm

Slit width: 0.7 nm

Lamp: Ag HCL, 10 mA

Background correction: Zeeman effect

Peak measurement: Peak area

Read delay: 0 sec

Read time: 5 sec

Output: To printer

GRAPHITE FURNACE SETTINGS:

Tube/platform: L’vov platform, pyrolytically coated

Carrier gas: Argon (high purity)

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas Read Step

Ramp Hold Flow (mL/min)

1 130 10 50 200

2 900 10 20 200

3 100 1 5 200

4 1400 0 5 0 *

5 2500 1 4 300

AUTOSAMPLER PROGRAM:

Solution: Volume (μL):

Sample 20

Blank 0

Modifier 1 10

Modifier 2 0

Number of injections: 1

Recalibrate after positions: 16, 28, and 40

STANDARDS: 0, 3, 6, and 9 ppb prepared from 1000 ppm Ricca standard in 0.2 M

HNO3.

46
MATRIX MODIFIERS: (1) 2.5% ammonium phosphate in 1 M HNO3;

 prepared from SPEX

ammonium dihydrogen orthophosphate

(2) none

APPROXIMATE SENSITIVITY: Approximately 0.130 A-sec for 20 μL of 3 ng/mL.

47
7.8. Arsenic

METHOD: Graphite furnace atomic absorption

INSTRUMENTATION:

Spectrophotometer: Perkin-Elmer 4100-ZL

Graphite furnace: Perkin-Elmer THGA

Autosampler: Perkin-Elmer AS 70

Printer: Okidata Microline 320

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelengths: 193.7 nm

197.0 nm (samples with high Al)

Slit width: 0.7 nm

Lamp: As EDL, 380 W

Background correction: Zeeman effect

Peak measurement: Peak area

Read delay: 0 sec

Read time: 5 sec

Output: To printer

GRAPHITE FURNACE SETTINGS:

Tube/platform: L’vov platform, pyrolytically coated

Carrier gas: Argon (high purity)

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas Read Step

Ramp Hold Flow (mL/min)

1 120 1 30 250

2 150 5 60 250

3 800 10 20 250

4 2200 0 5 0 *

5 2600 1 4 250

AUTOSAMPLER PROGRAM:

Solution: Volume (μL):

Sample 20

Blank 0

Modifier 1 10

Modifier 2 0

Number of injections: 1

Recalibrate after positions: 16, 28, and 40

48
STANDARDS: 0, 20, 40, and 60 ppb prepared from 1000 ppm Ricca standard in

0.2 M HNO3.

MATRIX MODIFIERS (1) 1:1:1 citric acid (2%): Pd (1000 ppm): Ni (4000 ppm)

(2) none

APPROXIMATE SENSITIVITY: Approximately 0.100 A-sec for 20 μL of 50 ng/mL at

193.7 nm; and 0.050 A-sec at 197.0 nm.

49
7.9. Cadmium

METHOD: Graphite furnace atomic absorption

INSTRUMENTATION:

Spectrophotometer: Perkin-Elmer Z3030

Graphite furnace: Perkin-Elmer HGA 600

Autosampler: Perkin-Elmer AS 60

Printer: Okidata Microline 184

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 228.8 nm

Slit width: 0.7 nm

Lamp: Cd EDL, 5 W

Background correction: Zeeman effect

Peak measurement: Peak area

Read delay: 0 sec

Read time: 5 sec

Output: To printer

GRAPHITE FURNACE SETTINGS:

Tube/platform: L’vov platform, pyrolytically coated

Carrier gas: Argon (high purity)

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas Read Step

Ramp Hold Flow (mL/min)

1 130 10 35 300

2 350 10 20 300

3 100 1 5 300

4 1300 0 5 0 *

5 2300 1 4 300

AUTOSAMPLER PROGRAM:

Solution: Volume (μL):

Sample 10

Blank 0

Modifier 1 5

Modifier 2 0

Number of injections: 1

Recalibrate after positions: 16, 28, and 40

STANDARDS: 0, 1, 2.5, and 4 ppb prepared from 1000 ppm Ricca standard in 0.2

M HNO3.

50
MATRIX MODIFIERS: (1) 2% citric acid

(2) none

APPROXIMATE SENSITIVITY: Approximately 0.110 A-sec for 10 μL of 1.4 ng/mL.

51
7.10. Chromium

METHOD: Graphite furnace atomic absorption

INSTRUMENTATION:

Spectrophotometer: Perkin-Elmer Z3030

Graphite furnace: Perkin-Elmer HGA 600

Autosampler: Perkin-Elmer AS 60

Printer: Okidata Microline 184

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 357.9 nm

Slit width: 0.7 nm

Lamp: Cr HCL, 25 mA

Background correction: Zeeman effect

Peak measurement: Peak area

Read delay: 0 sec

Read time: 5 sec

Output: To printer

GRAPHITE FURNACE SETTINGS:

Tube/platform: L’vov platform, pyrolytically coated

Carrier gas: Argon (high purity)

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas Read Step

Ramp Hold Flow (mL/min)

1 130 10 50 200

2 1000 10 20 200

3 100 1 5 200

4 2400 0 5 0 *

5 2500 1 4 300

AUTOSAMPLER PROGRAM:

Solution: Volume (μL):

Sample 15

Blank 0

Modifier 1 15

Modifier 2 0

Number of injections: 1

Recalibrate after positions: 16, 28, and 40

STANDARDS: 0, 7, 14, and 21 ppb prepared from 1000 ppm Ricca standard in

0.2 M HNO3.

52
MATRIX MODIFIERS: (1) Dilute NH3

, prepared in quartz-distilled water by isothermal

distillation

(2) none

APPROXIMATE SENSITIVITY: Approximately 0.160 A-sec with 15 μL of 7 ng/mL.

53
7.11. Copper

METHOD: Graphite furnace atomic absorption

INSTRUMENTATION:

Spectrophotometer: Perkin-Elmer Z3030

Graphite furnace: Perkin-Elmer HGA 600

Autosampler: Perkin-Elmer AS 60

Printer: Okidata Microline 184

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 324.8 nm

Slit width: 0.7 nm

Lamp: Cu HCL, 15 mA

Background correction: Zeeman effect

Peak measurement: Peak area

Read delay: 0 sec

Read time: 5 sec

Output: To printer

GRAPHITE FURNACE SETTINGS:

Tube/platform: L’vov platform, pyrolytically coated

Carrier gas: Argon (high purity)

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas Read Step

Ramp Hold Flow (mL/min)

1 110 10 35 200

2 500 10 20 200

3 100 1 5 200

4 1600 0 5 0 *

5 2500 1 4 300

AUTOSAMPLER PROGRAM:

Solution: Volume (μL):

Sample 10

Blank 0

Modifier 1 5

Modifier 2 0

Number of injections: 1

Recalibrate after positions: 16, 28, and 40

STANDARDS: 0, 20, 40, and 60 ppb prepared from 1000 ppm Ricca standard in

0.2 M HNO3.

54
MATRIX MODIFIERS: (1) Citric acid (2%)

(2) none

APPROXIMATE SENSITIVITY: Approximately 0.100 A-sec for 10 μL of 20 ng/mL.

55
7.12. Nickel

METHOD: Graphite furnace atomic absorption

INSTRUMENTATION:

Spectrophotometer: Perkin-Elmer Z3030

Graphite furnace: Perkin-Elmer HGA 600

Autosampler: Perkin-Elmer AS 60

Printer: Okidata Microline 184

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 232 nm

Slit width: 0.2 nm

Lamp: Ni HCL, 25 mA

Background correction: Zeeman effect

Peak measurement: Peak area

Read delay: 0 sec

Read time: 5 sec

Output: To printer

GRAPHITE FURNACE SETTINGS:

Tube/platform: L’vov platform, pyrolytically coated

Carrier gas: Argon (high purity)

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas Read Step

Ramp Hold Flow (mL/min)

1 130 10 40 200

2 1000 10 20 200

3 100 1 5 200

4 2200 0 4 0 *

5 2500 1 5 300

AUTOSAMPLER PROGRAM:

Solution: Volume (μL):

Sample 20

Blank 0

Modifier 1 5

Modifier 2 0

Number of injections: 1

Recalibrate after positions: 16, 28, and 40

STANDARDS: 0, 15, 30, and 45 ppb prepared from 1000 ppm Ricca standard in

0.2 M HNO3.

56
MATRIX MODIFIERS: (1) 2:2:1 Pd (1000 ppm) : La (1000 ppm) : MgNO3

 (2%)

(2) none

APPROXIMATE SENSITIVITY: Approximately 0.150 A-sec for 20 μL of 30 ng/mL.

57
7.13. Lead

METHOD: Graphite furnace atomic absorption

INSTRUMENTATION:

Spectrophotometer: Perkin-Elmer Z3030

Graphite furnace: Perkin-Elmer HGA 600

Autosampler: Perkin-Elmer AS 60

Printer: Okidata Microline 184

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 283.3 nm

Slit width: 0.7 nm

Lamp: Pb EDL, 10 W

Background correction: Zeeman effect

Peak measurement: Peak area

Read delay: 0 sec

Read time: 5 sec

Output: To printer

GRAPHITE FURNACE SETTINGS:

Tube/platform: L’vov platform, pyrolytically coated

Carrier gas: Argon (high purity)

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas Read Step

Ramp Hold Flow (mL/min)

1 130 10 45 200

2 800 10 15 200

3 100 1 5 200

4 1800 0 5 0 *

5 2500 1 5 300

AUTOSAMPLER PROGRAM:

Solution: Volume (μL):

Sample 20

Blank 0

Modifier 1 10

Modifier 2 0

Number of injections: 1

Recalibrate after positions: 16, 28, and 40

STANDARDS: 0, 10, 20, and 30 ppb prepared from 1000 ppm Ricca standard in

0.2 M HNO3.

58
MATRIX MODIFIERS: (1) 1:1 ammonium phosphate (4%, prepared from ammonium

dihydrogen orthophosphate) and citric acid (2%)

(2) none

APPROXIMATE SENSITIVITY: Approximately 0.160 A-sec for 20 μL of 30 ng/mL.

59
7.14. Selenium

METHOD: Graphite furnace atomic absorption

INSTRUMENTATION:

Spectrophotometer: Perkin-Elmer Z3030

Graphite furnace: Perkin-Elmer HGA 600

Autosampler: Perkin-Elmer AS 60

Printer: Okidata Microline 184

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 196.0 nm

Slit width: 0.7 nm

Lamp: Se EDL, 6 W

Background correction: Zeeman effect

Peak measurement: Peak area

Read delay: 0 sec

Read time: 5 sec

Output: To printer

GRAPHITE FURNACE SETTINGS:

Tube/platform: L’vov platform, pyrolytically coated

Carrier gas: Argon (high purity)

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas Read Step

Ramp Hold Flow (mL/min)

1 130 10 50 200

2 900 10 15 200

3 100 1 5 200

4 2300 0 5 0 *

5 2500 1 4 300

AUTOSAMPLER PROGRAM:

Solution: Volume (μL):

Sample 20

Blank 0

Modifier 1 10

Modifier 2 0

Number of injections: 1

Recalibrate after positions: 16, 28, and 40

STANDARDS: 0, 25, 50, and 75 ppb prepared from 1000 ppm Ricca standard in

0.2 M HNO3.

60
MATRIX MODIFIERS: (1) 5:3:1 Pd (1000 ppm) : Ni (4000 ppm) : hydroxylamine

hydrochloride (2%)

(2) none

APPROXIMATE SENSITIVITY: Approximately 0.140 A-sec for 20 μL of 50 ng/mL.

61
7.15. Tin

METHOD: Graphite furnace atomic absorption

INSTRUMENTATION:

Spectrophotometer: Perkin-Elmer Z3030

Graphite furnace: Perkin-Elmer HGA 600

Autosampler: Perkin-Elmer AS 60

Printer: Okidata Microline 184

ATOMIC ABSORPTION SPECTROPHOTOMETER SETTINGS:

Wavelength: 286.3 nm

Slit width: 0.7 nm

Lamp: Sn EDL, 8 W

Background correction: Zeeman effect

Peak measurement: Peak area

Read delay: 0 sec

Read time: 5 sec

Output: To printer

GRAPHITE FURNACE SETTINGS:

Tube/platform: L’vov platform, pyrolytically coated

Carrier gas: Argon (high purity)

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas Read Step

Ramp Hold Flow (mL/min)

1 130 10 50 200

2 800 10 10 200

3 100 1 5 200

4 2100 0 5 0 *

5 2300 1 4 300

AUTOSAMPLER PROGRAM:

Solution: Volume (μL):

Sample 20

Blank 0

Modifier 1 10

Modifier 2 0

Number of injections: 1

Recalibrate after positions: 16, 28, and 40

STANDARDS: 0, 10, 25, and 50 ppb prepared from 1000 ppm Ricca standard in

0.2 M HCl.

62
MATRIX MODIFIERS: (1) 1:1 magnesium nitrate (0.2%) and ammonium phosphate (4%,

prepared from ammonium dihydrogen orthophosphate)

(2) none

APPROXIMATE SENSITIVITY: Approximately 0.200 A-sec for 20 μL of 50 ng/mL.

63
7.16. Aluminum

METHOD: Instrumental neutron activation analysis for Al in sediments

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution germanium detector

Nuclear Data Genie MCA

IRRADIATION CONDITIONS:

Position: Pneumatics

Nominal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 30 sec

Cooling period: 15 min

Peak measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS:

Counting position: 2-10 cm

g-ray peak energy: 1779 KeV

Count time: 300 sec

STANDARDS:

Pure element standards: 0.01 g prepared from 1000 ppm Ricca standard.

Matrix standards: 0.1 g of: TAMU HS2* , USGS GXR-5, NIST SRM 1646, NRC

BCSS-1, NRC MESS-2.

SAMPLE PREPARATION: Approximately 0.1 g weighed to nearest 0.0001 g into 0.4-

dram polyethylene vials.

APPROXIMATE SENSITIVITY: Depends upon background; 100 μg results in approximately

1000 counts with associated counting error of 10%; with

typical background and 0.1 g sample size, this corresponds

to 0.1% Al.

*

 TAMU HS2 is a "house" reference sediment standard collected using a box corer in the Mississippi river delta. The sediment

was washed several times in distilled water to remove dissolved salts, freeze dried, ground with a mortar and pestle, and

homogenized.

64
7.17. Chromium

METHOD: Instrumental neutron activation analysis for Cr in sediments

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution germanium detector

Nuclear Data Genie MCA

IRRADIATION CONDITIONS:

Position: Rotisserie

Nominal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 14 hr

Cooling period: 10 days

Peak measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS:

Counting position: 10 cm

g-ray peak energy: 320.1 KeV

Count time: 60 min

STANDARDS:

Pure element standards: 1000 μg prepared from 1000 ppm Ricca standard.

Matrix standards: 0.5 g of: TAMU HS2, USGS GXR-5, NIST SRM 1646, NRC

BCSS-1, NRC MESS-2.

SAMPLE PREPARATION: Approximately 0.5 g weighed to nearest 0.0001 g into 0.4-

dram polyethylene vials.

APPROXIMATE SENSITIVITY: Depends upon background; 1 μg results in approximately

1000 counts with associated counting error of 15%; with

typical background and 0.5 g sample size, this corresponds

to 2 ppm Cr.

65
7.18. Iron

METHOD: Instrumental neutron activation analysis for Fe in sediments

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution germanium detector

Nuclear Data Genie MCA

IRRADIATION CONDITIONS:

Position: Rotisserie

Nominal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 14 hr

Cooling period: 10 days

Peak measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS:

Counting position: 10 cm

g-ray peak energy: 1099.2 KeV

Count time: 60 min

STANDARDS:

Pure element standards: 0.01 g prepared from 1000 ppm Ricca standard.

Matrix standards: 0.5 g of: TAMU HS2, USGS GXR-5, NIST SRM 1646, NRC

BCSS-1, NRC MESS-2.

SAMPLE PREPARATION: Approximately 0.5 g weighed to nearest 0.0001 g into 0.4-

dram polyethylene vials.

APPROXIMATE SENSITIVITY: Depends upon background; 150 μg results in approximately

1000 counts with associated counting error of 10%; with

typical background and 0.5 g sample size, this corresponds

to 300 ppm Fe.

66
7.19. Manganese

METHOD: Instrumental neutron activation analysis for Mn in sediments

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution germanium detector

Nuclear Data Genie MCA

IRRADIATION CONDITIONS:

Position: Pneumatics

Nominal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 30 sec

Cooling period: 15 min

Peak measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS:

Counting position: 2-10 cm

g-ray peak energy: 846.8 KeV

Count time: 300 sec

STANDARDS:

Pure element standards: 1000 μg prepared from 1000 ppm Ricca standard.

Matrix standards: 0.1 g of TAMU HS2, USGS GXR-5, NIST SRM 1646, NRC

BCSS-1, NRC MESS-2.

SAMPLE PREPARATION: Approximately 0.1 g weighed to nearest 0.0001 g into 0.4-

dram polyethylene vials.

APPROXIMATE SENSITIVITY: Depends upon background: 1 μg results in approximately

1000 counts with associated counting error of 10%; with

typical background and 0.1 g sample size, this corresponds

to 10 ppm Mn.

67
7.20. Arsenic

METHOD: Instrumental neutron activation analysis for As in tissues

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution, high-purity germanium detector

Nuclear Data Genie MCA implemented on Digital VAX workstation

IRRADIATION CONDITIONS:

Position: Rotisserie

Nominal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 14 hr

Cooling period: 7 days

Peak measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS:

Counting position: Variable, depending upon sample activity; normally 10-

20 cm

Gamma-ray peak energy: 559.1 KeV

Count time 30-45 min, depending on concentration

STANDARDS:

Pure element standards: 6 μg prepared from 1000 ppm Ricca standard

Matrix standards: 0.25 g of: NRC DOLT-2; NRC DORM-2, NIST SRM

1566

SAMPLE PREPARATION: Approximately 0.25 to 0.5 g tissue weighed to

nearest 0.0001 g into 0.4 dram polyethylene vial

APPROXIMATE SENSITIVITY: Depends upon background; 0.85 μg, results in

approximately 1000 counts above background with

associated counting error of 10%; with typical background

and 0.25 g sample size, this corresponds to 3.4 ppm on a

dry weight basis.

68
7.21. Chromium

METHOD: Instrumental neutron activation analysis for Cr in tissues

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution, high-purity germanium detector

Nuclear Data Genie MCA implemented on Digital VAX workstation

IRRADIATION CONDITIONS:

Position: Rotisserie

Nominal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 14 hr

Cooling period: 10 days

Peak measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS.

Counting position: 1-10 cm

Gamma-ray peak energy: 320.1 KeV

Count time: 75 min

STANDARDS:

Pure element standards: 5 -g prepared from 1000 ppm Ricca standard

Matrix standards: 0.25 g of: NRC DOLT-2; NRC DORM-2, NIST SRM

1566a.

SAMPLE PREPARATION: Approximately 0.25 to 0.5 g tissue weighed to

nearest 0.0001 g into 0.4 dram polyethylene vial

APPROXIMATE SENSITIVITY: Depends upon background; 0.25 μg, results in

approximately 1000 counts above background with

associated counting error of 10%; with typical

background and 0.25 g sample size, this

corresponds to 1.0 ppm Cr on a dry weight basis.

69
7.22. Iron

METHOD: Instrumental neutron activation analysis for Fe in tissues

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution, high-purity germanium detector

Nuclear Data Genie MCA implemented on Digital VAX workstation

IRRADIATION CONDITIONS:

Position: Rotisserie

Nominal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 14hr

Cooling period: 10 days

Peak measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS:

Counting position: 1-10 cm

Gamma-ray peak energy: 1099.2 KeV

Count time: 75 min

STANDARDS:

Pure element standards: 333 μg prepared from 1000 ppm Ricca standard

Matrix standards: 0.25 g of: NRC DOLT-2; NRC DORM-2, NIST SRM

1566a

SAMPLE PREPARATION: Approximately 0.25 to 0.5 g tissue weighed to nearest

0.0001 g into dram polyethylene vial

APPROXIMATE SENSITIVITY: Depends upon background; 36 μg, results in approximately

1000 counts above background with associated counting

error of 10%; with typical background and 0.25 sample

size, this corresponds to 145 ppm on a dry weight basis.

70
7.23. Selenium

METHOD: Instrumental neutron activation analysis for Se in tissues

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution, high-purity germanium detector

Nuclear Data Genie MCA implemented on Digital VAX workstation

IRRADIATION CONDITIONS:

Position: Rotisserie

Nominal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 14 hr

Cooling period: 10 days

Peak measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS:

Counting position: 1-10 cm

Gamma-ray peak energy: 264.6 KeV

Count time: 75 min

STANDARDS:

Pure element standards 6 μg prepared from 1000 ppm Ricca standard

Matrix standards: 0.25 g of: NRC DOLT-2; NRC DORM-2, NIST SRM

1566a

SAMPLE PREPARATION: Approximately 0.25 to 0.5 g tissue weighed to

nearest 0.0001 g into 0.4 dram polyethylene vial

APPROXIMATE SENSITIVITY: Depends upon background; 0.22 μg, results in

approximately 1000 counts above background

associated counting error of 10%; with typical

background and 0.25 g sample size, this

corresponds to 0.87 ppm on a dry weight basis.

71
7.24. Silver

METHOD: Instrumental neutron activation analysis for Ag in tissues

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution, high-purity germanium detector

Nuclear Data Genie MCA implemented on Digital VAX workstation

IRRADIATION CONDITIONS:

Position: Rotisserie

Nominal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 14 hr

Cooling period: 10 days

Peak measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS:

Counting position: 1-10 cm

Gamma-ray peak energy: 657.8 KeV

Count time: 75 min

STANDARDS:

Pure element standards: 5 μg prepared from 1000 ppm Ricca standard

Matrix standards: 0.25 g of: NRC DOLT-2; NRC DORM-2, NIST SRM

1566a

SAMPLE PREPARATION: Approximately 0.25 to 0.5 g tissue weighed to

nearest 0.0001 g into 0.4 dram polyethylene vial

APPROXIMATE SENSITIVITY: Depends upon background; 0.14 μg, results in

approximately 1000 counts above background with

associated counting error of 10%; with typical

background and 0.25 g sample size, this

corresponds to 0.55 ppm Ag on a dry weight basis.

72
7.25. Zinc

METHOD: Instrumental neutron activation analysis for Zn in tissues

INSTRUMENTATION:

TRIGA 1 MW reactor

Ortec high-resolution, high-purity germanium detector

Nuclear Data Genie MCA implemented on Digital VAX workstation

IRRADIATION CONDITIONS:

Position: Rotisserie

Normal neutron flux: 1 x 1013 neutrons/cm2/sec

Length of irradiation: 14 hr

Cooling period: 10 days

Peak Measurement: Net peak area using Nuclear Data peak program

COUNTING CONDITIONS:

Counting position: 1-10 cm

Gamma-ray peak energy: 1115.6 KeV

Count time: 75 min

STANDARDS:

Pure element standards: 10 μg prepared from 1000 ppm Ricca standard

Matrix standards: 0.25 g of: NRC DOLT-2, NRC-DORM-2, NIST SRM

1566A

SAMPLE PREPARATION: Approximately 0.25 to 0.5 g tissue weighed to

nearest 0.0001 g into 0.4 dram polyethylene vial

APPROXIMATE SENSITIVITY: Depends upon background; 2.3 μg, results in

approximately 1000 counts above background with

associated counting error of 10%; with typical

background and 0.25 g sample size, this

corresponds to 9.1 ppm Zn on a dry weight basis.

73
Analysis of Marine Sediment and Bivalve Tissue

by X-Ray Fluorescence, Atomic Absorption and Inductively Coupled

Plasma Mass Spectrometry

E. Crecelius, C. Apts, L. Bingler, J. Brandenberger, M. Deuth, S. Kiesser, and R. Sanders *

Battelle/Marine Sciences Laboratory

1529 West Sequim Bay Rd.

Sequim, Washington

ABSTRACT

Analytical chemistry techniques including atomic absorption spectrophotometry,

inductively coupled plasma mass spectrometry (ICP-MS) and energy dispersive x-ray

fluorescence (XRF) have been applied to the analysis of 17 elements in aquatic sediment

and bivalve tissue in support of the NOAA NS&T Program. Complete acid digestion of

samples at elevated pressure and temperature in a sealed Teflon container minimizes

contamination and loss of elements. Multi-elemental techniques such as ICP-MS and XRF

provide sensitive, accurate, and precise results for a variety of elements at a

reasonable cost.

1. INTRODUCTION

Methods used for analysis of 17 metals in estuarine sediments and tissues were developed as

part of the Mussel Watch Project of the National Status and Trends (NS&T) Program. The total

digestion procedure used for sediments, without loss of volatile elements, was developed by

Taylor and Presley (this volume) at Texas A&M University (TAMU). The digestion procedure

for tissues was a combination of HCl and HNO3. Metals were analyzed predominantly by

graphite furnace atomic absorption (GFAA) spectrometry and x-ray fluorescence (XRF).

Mercury was analyzed by cold vapor atomic absorption (CVAA). Selected metals were

determined by inductively-coupled plasma mass spectrometry (ICP-MS). The primary

objective in developing each instrumental method was to keep the method as simple and

straightforward as possible while producing acceptable accuracy and precision.

It is expected that users of the instrumental methods described below will modify certain

parameters to suit their particular instrumentation and equipment due to variations in

performance between instruments. Such variations are caused by differences in furnace

calibration, lamp intensities, nebulizer characteristics, and other parameters.

2. EQUIPMENT AND SUPPLIES

2.1. Instrumentation

Perkin-Elmer model Z/5000 spectrophotometer. Perkin-Elmer, Norwalk, CT.

Dual lamp EDL power supply

Model 500 graphite furnace atomizer with Zeeman background correction system

Model 3600 data terminal

Model AS40 autosampler

*

 KLM Analytical, 2000 Logston Blvd., Richland, WA 99352.

74
Perkin-Elmer model Z/3030 spectrophotometer

Dual lamp EDL power supply

Model 600 graphite furnace atomizer with Zeeman background correction system

Model AS60 autosampler

Model PRS 100 printer

Thermo-Separation Products Fully Automated Mercury Analysis System, MM3200, TSP,

Portland, OR.

KEVEX x-ray fluorescence excitation and detection subsystem, 0810Z. Fisons Instruments,

San Carlos, CA.

Canberra series 80 multichannel analyzer. Canberra Nuclear Products, Merider, CT.

Digital PDP-11/34A computer operating system

Digital RL01 floppy disc drive

Digital RL01 hard disc drive

Digital video terminal (model 102)

KEVEX 4620 detector bias supply

KEVEX high voltage generator

PHA/LTC model 8623 amplifier

Versatec printer/plotter. Versatec Inc., Santa Clara, CA.

Perkin-Elmer model 5000 inductively coupled plasma mass spectrometer

2.2. Supplies

Argon, 99.999% purity Hollow cathode lamps (HCL)

Electrodeless discharge lamps (EDL) Laboratory press, 3.2-cm diameter, 27,000

Electron multiplier, model 4870V, Galileo kg

channeltron. Galileo Inc., Sturbridge,

MA.

Nitrogen, ultra-pure

Platforms, pyrolytically coated L'vov,

Graphite tubes, pyrolytically coated, Perkin-Elmer B0121-091

grooved, Perkin-Elmer B0121-092.

Perkin-Elmer, Norwalk, CT.

Quartz torch

Sample cones, nickel

Graphite tubes, pyrolytically coated, nongrooved, Perkin-Elmer B0135-653.

Perkin-Elmer, Norwalk, CT.

Skimmer cones, nickel

Thin film standards. Micro Matter,

Eastsound, WA

Helium

2.3. Labware

Balance, Mettler model AC100 and H30,

Sartorius MCI LAB LC1200 S

Flask, volumetric, polyethylene, 100-mL

Freeze dryers, 6206-0101. Virtis Co.,

Bomb lids, 120-mL, Teflon perfluoralkoxy Gardiner, NY.

(PFA), double-ported, 0104-4-2.

Savillex, Minnetonka, MN.

Jars, 125-mL, polystyrene, 8002. Spex

Industries, Edison, NJ.

Bombs, 60-mL, Teflon perfluoralkoxy (PFA)

bomb, 561R2; 120-mL, Teflon

perfluoralkoxy (PFA), 577. Savillex,

Minnetonka, MN.

Methacrylate balls, 3112. Spex Industries,

Edison, NJ.

Mixer/Mills, model 8000. Spex Industries,

Edison, NJ.

Capping station. CEM Corp., Mathews, NC. Oven, stainless steel, Imperial II Radiant

Heat Oven. Lab Line Inc., Melrose Park,

IL.

Cups, 2-mL, polystyrene, B2713-2. Baxter,

McGaw Park, IL.

75
Pipettes, macro and micro Teflon tubing, 1-mm i.d.

Polyethylene tubing, 7420. Clay Adams Co.,

Parsippany, NJ.

Vials, 20-mL, polyethylene, threaded, with

screw caps, Kimble 66022-241

Spatulas Vials, dry grinding, ceramic, 8003. Spex

Stopwatch or timer Industries, Edison, NJ.

Polyethylene vials were cleaned by soaking for five days in 5% HNO3, at room temperature,

rinsed with deionized water and dried in Class 100 laminar flow hoods. All other plastic ware

was soaked for three days in 10% HNO3 at room temperature, rinsed with deionized water,

and dried as above. Teflon bombs were soaked for two days in 50% concentrated HNO3.

2.4. Reagents

All reagents are ultrapure grades except where so designated.

Ammonium dihydrogen phosphate (NH4H2PO4)

[7722-76-1], Ultrex 7-9431. J. T.

Baker, Phillipsburg, NJ.

Nickel nitrate [Ni(NO3)2 

. 6H2O] [13478-00-

7], solid, Puratonic Grade. Johnson

Matthey Chemicals, West Chester, PA.

Ammonium nitrate (NH4NO3) [6484-52-2],

,

solid, reagent grade, 729-1. J. T. Baker,

Phillipsburg, NJ.

Nitric acid (HNO3) [7697-37-2], concentrated (70%), Instra-analyzed, 9598-

33. J. T. Baker, Phillipsburg, NJ.

Ascorbic acid (C6H8O6) [50-81-7], solid,

reagent grade, 581-5. J. T. Baker,

Phillipsburg, NJ.

Perchloric acid (HClO4) [7601-90-3],

concentrated (70%), 230. G. Frederick

Smith, Columbus, OH.

Atomic absorption standards, 1000 μg/mL.

High-Purity Standards, Inc., Charleston,

SC.

Soda lime [variable mixture of NaOH and

CaO/Ca(OH)2] [8006-28-8], 4-8 mesh,

used as acid fume trap in CVAA, 3448-

01. J. T. Baker, Phillipsburg, NJ. Hydrochloric acid (HCl) [7647-01-0],

concentrated (37%), Instra-analyzed,

9530-33. J. T. Baker, Phillipsburg, NJ.

Stannous chloride (SnCl2

. 2H2O) [10025-

69-1], 3980-01. J. T. Baker,

Phillipsburg, NJ. Hydrofluoric acid (HF) [7664-39-3], Instraanalyzed, 9563-01. J. T. Baker,

Phillipsburg, NJ. Sulfuric acid (H2

SO4) [7664-93-9], concentrated (98%), Instra-analyzed,

9673-33. J. T. Baker, Phillipsburg, NJ. Magnesium nitrate [Mg(NO3

)2

. 6H2O]

[13446-18-9], solid, Puratonic Grade.

Johnson Matthey Chemicals, West

Chester, PA.

Water, deionized, 10 megohm-cm resistivity.

2.5. Solvents and matrix modifiers

Ammonium phosphate: 2%, monobasic, 2 g of NH4H2PO4 and 0.200 g of Mg(NO3)2per liter

of deionized water.

Ammonium phosphate: 4%, monobasic, 4 g NH4H2PO4 and 13 g NH4NO3 diluted to 100 mL

with deionized water.

Magnesium nitrate solution: 250 mg/L of deionized water.

0.10 M Nickel nitrate: 2.91 g Ni(NO3)2

.6H2O in 100 mL of deionized water.

Sulfuric acid modifier solution: Dilute 1 mL of concentrated sulfuric acid to 99 mL with

deionized water and add 10 μL of 0.1 M Ni(NO3)2

.6H2O.

76
3. SAMPLE TREATMENT

3.1. Drying and homogenization

3.1.1. Sediments

Sediments were obtained with a grab sampler that collects the top 2 cm of surface sediment.

Sample aliquots were weighed, freeze-dried for approximately 5 days to constant weight in

125-mL polystyrene Spex jars in a freeze dryer, and reweighed to determine percent dry

weight. Approximately 3 g were ground for 5 min using a Spex ceramic ball mill. Aliquots of

0.5 g were used for X-ray fluorescence, or digested for atomic absorption or ICP-MS

analysis.

3.1.2. Tissues

Bivalves were hand-collected and shucked at the lab. Tissues were freeze-dried in Spex jars

and homogenized using methacrylate balls in a Spex mixer/mill.

3.2. Digestion

Battelle used minor modifications of the total digestion techniques for sediments developed by

Taylor and Presley (this volume). Tissues were digested with a mixture of HCl and HNO3

which has been shown to provide much better recovery of Ag than HNO3 or HNO3and HClO4

(Crecelius and Daskalakis, 1994; Daskalakis et al., 1997).

Two reagent blanks and three standard reference material samples were included in each

analytical string of 50 samples. Reagent blanks contained no sediment or tissue and were

processed like the samples.

3.2.1. Sediments

Approximately 200 ± 7 mg of homogenized ground dried sediment was weighed into a tared

60-mL Teflon digestion bomb. One mL of 4:1 HNO3/HClO4 was added to each bomb and the

lid tightened at the capping station.

The bombs were heated in the oven at 130 ± 10 °C for 4 hr and allowed to cool.

Three mL of concentrated HF were added to each bomb, rinsing the walls of the bomb to

insure that all solids were washed down into the acid mixture. The lid was tightened at the

capping station.

The bombs were heated in the oven at 130 ± 10 °C for 8 hr and allowed to cool.

The bombs were opened and the digestates diluted to approximately 20 mL with deionized

water.

The solutions were weighed in the Teflon bombs and the volume calculated using a density

factor of 1.05 g/mL.

The contents of each bomb were transferred quantitatively to a 20-mL polyethylene

screw-cap vial for storage prior to analysis.

77
Digestates were analyzed directly by GFAA or CVAA. Before analysis by ICP-MS, a 10-mL

aliquot was transferred to a Teflon bomb and dried slowly, uncovered, on a hot plate (203 °C)

in a perchloric acid hood to eliminate chloride and fluoride. White fumes were indicative of

successful elimination of these elements. The dried digestate was dissolved in 1 mL of 10%

HNO3 and heated again to dryness. The dried digestate was dissolved again with 1 mL of 10%

HNO3 and 9 mL of deionized water.

3.2.2. Tissues

Approximately 500 ± 15 mg of homogenized, dried tissue was weighed into a tared 60-mL

Teflon digestion bomb. Five mL of HCl and 3.5 mL of HNO3 were added to each bomb and the

lid screwed on but not tightened.

The bombs were placed in a cold water bath in an acid hood and the water bath was heated

slowly to 60 ± 3 °C. The total heating time was 3 to 4 hr.

The bombs were allowed to cool. The lids were tightened at the capping station.

The bombs were heated in the oven at 130 ± 10 °C for 16 hr and allowed to cool.

The bombs were opened and the digestates diluted to approximately 20 mL with deionized

water.

The solutions were weighed in the Teflon bombs and the volume calculated using a density

factor of 1.09 g/mL.

Digestates were analyzed directly by GFAA and CVAA, or diluted 10:1 for ICP-MS analysis.

4. CALIBRATION

Calibration standards were prepared by serial dilution of commercially available atomic

absorption standards using class-A glass pipettes, volumetric flasks, and 10% HNO3. Some

metals, such as Sb and Sn, required use of a different acid diluent. Final working standards

were prepared in 1% HNO3 or the appropriate acid diluent, using micropipettes and glass

volumetric flasks. The element concentrations in each standard should be sufficient to cover

the appropriate range of sample concentrations and produce good measurement precision and

accurately define the slope of the response curve. Concentrations of commercial standards

were verified by comparison with National Institute of Standards and Technology (NIST)

spectrophotometric standards.

5. SPECTRAL INTERFERENCES

Spectral interferences may be minimized by sample dilution, use of an alternate analyte

wavelength, or selective volatilization of the analyte. Non-spectral interferences may be

detected and compensated for using the method of standard additions. Matrix modifiers that

were used include Mg(NO3)2, NH4H2PO4, Ni(NO3)2, and H2SO4. Their specific use is described

in the instrumental analysis section for each element.

78
6. CALCULATIONS

6.1. Graphite furnace and ICP-MS

Sample concentrations were determined from calibration results and from the dilution factors

using the following equation:

Concentration (μg/g dry wt) = 

(SA - B A) m S V

1000 (SDW) (DF)

where SA was the sample absorbance or intensity value, BA was the procedural blank

absorbance or intensity value, m was the slope of standard addition calibration line, SV was

the sample volume, SDW was the sample dry weight in grams, and DF was the dilution factor.

6.2. Cold vapor atomic absorption

Sample concentrations were determined from calibration results and from the dilution factors

involved in instrumental analysis and sample digestion according to the following equation:

Concentration (μg/g dry wt) = 

(SA - B A) m S V

1000 (SDW) (DF)

where SA was the sample absorbance value, BA was the procedural blank absorbance value, m

was the slope of the calibration line, SV was the sample volume, SDW was the sample dry

weight in grams, and DF was the dilution factor.

6.3. X-ray fluorescence

This procedure used energy dispersive x-ray fluorescence spectroscopy to quantify elemental

concentrations in sediment and tissue samples (Nielson et al., 1982). The backscatter/

fundamental parameter approach using the SAP3 computer code incorporated thin film

standards and scattering rations to produce matrix corrections (Nielson and Sanders, 1982).

Thin film standards were used for the determination of intensity in count/min/μg

(element)/cm2 versus element energy in KeV. Thin film standards produced by vapor deposit

of the elements on Mylar or polycarbonate substrate were purchased from Micro Matter of

Eastsound, WA. These standards were traceable to the U.S. National Institute of Standards and

Technology.

The peak analysis and elemental concentration sections of the computer program (SAP3) used

arrays of fundamental physical parameters of x-ray energies, mass adsorption coefficients,

cross sections, fluorescence yields, absorption edge, and jump ratios to perform the matrix

corrections for relating net peak intensities to element concentrations. These arrays were

read into the computer from a disc file when the program was initiated. Each excitation source

had its own unique disc file or library. Thin film sensitivities or calibration factors of the

spectrometer using the intended excitation source was also part of the library.

79
7. CONCLUSIONS

A variety of analytical instruments provided the optimum method for the analysis of 17

elements in sediment and bivalve tissue. The advantage of XRF was that the sample did not

require digestion but was analyzed as a dry powder. Crustal elements such as Al, Cr, Fe, Ni,

and Si, that are difficult to dissolve from sediment, could be quantified by XRF. Also, Se and

As can be difficult to quantify in tissue digestates by ICP-MS but were quantified easier using

XRF. ICP-MS had the advantage of simultaneous analysis of many elements with detection

limits much lower than the XRF and similar to those of GFAA. Elements that were particularly

sensitive and relatively interference free by ICP-MS include Al, Cr, Ni, Ag, Cd, Sn, Sb, Pb,

and Tl. Cold vapor atomic absorption is a very sensitive and reliable technique for Hg analysis.

Care must be taken to avoid leakage at high pressure or Hg can be lost during digestion. With

the use of the sealed Teflon digestion vessel, mercury could be analyzed from the same

digestion as the other metals (Al, Cr, Ni, Ag, Cd, Sn, Sb, Pb, and Tl).

The advantage of freeze drying both sediment and tissue was that the dry material was easily

ground or homogenized. No Hg loss occurred during freeze drying. Sediment digestates

received special treatment to remove silicon, fluoride and perchloric acid before analysis by

ICP-MS. Analysis by GFAA requires matrix modifiers and standardization of the instrument by

method of addition to the sample matrix to provide accurate results.

8. REFERENCES

Crecelius, E. A., and K. D. Daskalakis (1994) Analysis of silver in mussels and oysters by

inductively coupled plasma mass spectrometry (ICPMS). Proc., Second Internatl. Conf.,

Transport, Fate and Effects of Silver in the Environment. A. W. Andren and T. W. Bober (eds.).

September 11-13, 1994. Madison, WI. 157-9.

Daskalakis, K. D., T. P. O'Connor, and E. A. Crecelius (1997) Evaluation of digestion

procedures for determining silver in mussels and oysters. Environ. Sci. Technol., 31:2303-

2306).

Nielson, K. K., and R. W. Sanders (1982) The SAP3 computer program for quantitative multielement analysis by energy-dispersive x-ray fluorescence. PNL-4173, Battelle Pacific

Northwest Laboratory, Richland, WA. 120 pp.

Nielson, K. K., R. W. Sanders, and J. C. Evans (1982) Analysis of steels by energy-dispersive

x-ray fluorescence with fundamental parameters. Anal. Chem., 54(11):1782-6.

Patterson, K. Y., C. Veillon, and H. M. Kingston (1988) Microwave digestion of biological

samples. In: Introduction to Microwave Sample Preparation. H. M. Kingston and L. B. Jassie

(eds.). 155-66. ACS Professional Reference Book, American Chemical Society, Washington,

DC.

80
9. INSTRUMENTAL ANALYSIS

9.1. Atomic absorption spectrometry

9.1.1. Aluminum

Graphite furnace atomic absorption for tissue

METHOD: Graphite furnace atomic absorption

DIGEST MATRIX: ~15% 4:1 HNO3:HClO4 by volume.

INSTRUMENT SETTINGS:

Wavelength: 309.3 nm

Lamp: HCL, 25 ma (Perkin-Elmer 0303-6009)

Tube: Non-platform, pyrolytically coated

Carrier gas: Argon

Slit width: 0.7

Background correction: Zeeman

Signal mode: Peak height

Scale expansion: None

Read time: 5 sec

Output: Recorded from spectrophotometer display

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas

Ramp Hold Flow (mL/min)

Dry 1 80 9 1 300

Dry 2 140 45 5 300

Dry 3 250 20 5 300

Char 1600 45 25 300

Atomize 2300 0 4 50

Cleanout 2650 1 3 300

Cool 20 1 10 300

STANDARDS: Addition calibration using 22.2, 43.5, and 83.3 μg/L Al to CRM

DOLT-1 tissue digestate. Method of standard addition.

TYPICAL SENSITIVITY: Absorbance is approximately 0.100 for 0.4 μg Al standard.

CALIBRATION: Peak height versus concentration of standards used to compute the

slope, intercept, and correlation coefficient of calibration line

using linear, least-squares regression.

INJECTION VOLUME: 5 μL

MATRIX MODIFIER: 5 μL of 2% NH4H2PO4.

81
9.1.2. Chromium

Graphite furnace atomic absorption for tissue

METHOD: Graphite furnace atomic absorption

DIGEST MATRIX: ~15% 4:1 HNO3:HClO4 by volume.

INSTRUMENT SETTINGS:

Wavelength: 357.9 nm

Lamp: HCL, 25 ma (Perkin-Elmer 0303-6021)

Tube: Pyro, non-platform

Carrier gas: Argon

Slit width: 0.7

Background correction: Zeeman

Signal mode: Peak height

Scale expansion: None

Read time: 5 sec

Output: Recorded from spectrophotometer display

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas

Ramp Hold Flow (mL/min)

Dry 1 80 9 1 300

Dry 2 140 45 5 300

Dry 3 250 20 5 300

Char 1500 20 25 300

Atomize 2500 0 4 0

Cleanout 2650 1 3 300

Cool 20 1 10 300

STANDARDS: Method of standard addition calibration using 20.0, 33.3, and 66.7

μg/L Cr to CRM DOLT-1 tissue digestate.

TYPICAL SENSITIVITY: Absorbance is approximately 0.200 for 0.165 μg Cr standard.

CALIBRATION: Peak height versus concentration of standards used to compute the

slope, intercept, and correlation coefficient of calibration line

using linear, least-squares regression.

INJECTION VOLUME: 5 μL

MATRIX MODIFICATION: 5 μL of 250 mg/L Mg as Mg(NO3)2.

82
9.1.3. Nickel

Graphite furnace atomic absorption for tissues

METHOD: Graphite Furnace Atomic Absorption

DIGEST MATRIX: ~15% 4:1 HNO3:HClO4 by volume.

INSTRUMENT SETTINGS:

Wavelength: 232.0 nm

Lamp: HCL, 35 ma (Perkin-Elmer 0303-6047)

Tube: Pyro, non-platform

Carrier gas: Argon

Slit width: 0.2

Background correction: Zeeman

Signal mode: Peak height

Scale expansion: None

Read time: 5 sec

Output: To printer

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas

Ramp Hold Flow (mL/min)

Dry 1 80 9 1 300

Dry 2 140 45 5 300

Dry 3 250 20 5 300

Char 1200 20 20 300

Atomize 2300 0 4 0

Cleanout 2650 1 3 300

Cool 20 1 10 300

STANDARDS: Method of standard addition calibration using 24.39, 47.62, and

90.91 μg/L Ni to CRM DOLT-1 digestate.

TYPICAL SENSITIVITY: Absorbance is approximately 0.100 for 0.6 μg Ni standard.

CALIBRATION: Peak height of standard versus concentration of standard. Compute

the slope, intercept, and correlation coefficient using linear,

least-squares regression.

INJECTION VOLUME: 5 μL

MATRIX MODIFICATION: 5 μL of 2% NH4H2PO4.

MODIFIER VOLUME: 5 μL

83
9.1.4. Selenium

Graphite furnace atomic absorption for sediment

METHOD: Graphite Furnace Atomic Absorption

DIGEST MATRIX: ~4% 4:1 HNO3:HClO4 + 12% HF by volume.

INSTRUMENT SETTINGS:

Wavelength: 195.9 nm

Lamp: EDL, 6 watts (Perkin-Elmer 0303-6262)

Tube: L’vov, pyro coated

Carrier gas: Argon

Slit width: 2.0

Background correction: Zeeman

Signal mode: Peak height

Scale expansion: None

Read time: 5 sec

Output: Recorded from spectrophotometer display

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas

Ramp Hold Flow (mL/min)

Dry 1 80 9 1 300

Dry 2 140 45 5 300

Dry 3 250 20 5 300

Char 1300 20 20 300

Atomize 2100 0 4 0

Cleanout 2650 1 3 300

Cool 20 1 10 300

STANDARDS: Method of standard addition calibration using 47.6, 90.9, and

130.4 μg/L Se to CRM MESS-1 sediment digestate.

TYPICAL SENSITIVITY: Absorbance is approximately 0.100 for 1.0 μg Se standard.

CALIBRATION: Peak height of standard versus concentration of standard. Compute

the slope, intercept, and correlation coefficient using linear,

least-squares regression.

INJECTION VOLUME: 20 μL

MATRIX MODIFICATION: 10 μL of 0.1 M Ni(NO3)2.

84
9.1.5. Silver

9.1.5.1. Graphite furnace atomic absorption for tissue

METHOD: Graphite Furnace Atomic Absorption

DIGEST MATRIX: ~15% 4:1 HNO3:HClO4by volume.

INSTRUMENT SETTINGS:

Wavelength: 327.9 nm

Lamp: HCL, 12 ma (Perkin-Elmer 0303-6064)

Tube: L’vov, pyro coated

Carrier gas: Argon

Slit width: 0.7

Background correction: Zeeman

Signal mode: Peak height

Scale expansion: None

Read time: 4 sec

Output: To printer

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas

Ramp Hold Flow (mL/min)

Dry 1 80 9 1 300

Dry 2 140 45 5 300

Dry 3 250 20 5 300

Char 800 20 20 300

Atomize 2100 0 4 0

Cleanout 2650 1 3 300

Cool 20 1 10 300

STANDARDS: Addition calibration using 0.99, 1.96, and 4.76 μg/L Ag to CRM

DOLT-1 tissue digestate.

TYPICAL SENSITIVITY: Absorbance is approximately 0.100 for 0.03 μg Ag standard.

CALIBRATION: Peak height of standard versus concentration of standard. Compute

the slope, intercept, and correlation coefficient using linear,

least-squares regression.

INJECTION VOLUME: 10 μL

MATRIX MODIFICATION: 5 μL of 2% NH4H2PO4.

85
Silver

9.1.5.2. Graphite furnace atomic absorption for sediment

METHOD: Graphite Furnace Atomic Absorption

DIGEST MATRIX: ~4% 4:1 HNO3:HClO4 +12% HF by volume.

INSTRUMENT SETTINGS:

Wavelength: 327.9 nm

Lamp: HCL, 12 ma (Perkin-Elmer 0303-6064)

Tube: L’vov, pyro coated

Carrier gas: Argon

Slit width: 0.7

Background correction: Zeeman

Signal mode: Peak height

Scale expansion: None

Read time: 4 sec

Output: To printer

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas

Ramp Hold Flow (mL/min)

Dry 1 80 9 1 300

Dry 2 140 45 5 300

Dry 3 250 20 5 300

Char 800 20 20 300

Atomize 2100 0 4 0

Cleanout 2650 1 3 300

Cool 20 1 10 300

STANDARDS: Addition calibration using 2.44, 4.76, and 9.09 μg/L Ag to SRM

1646 sediment digestate.

TYPICAL SENSITIVITY: Absorbance is approximately 0.120 for 0.05 μg Ag standard.

CALIBRATION: Peak height of standard versus concentration of standard. Compute

the slope, intercept, and correlation coefficient using linear,

least-squares regression.

INJECTION VOLUME: 10 μL

MATRIX MODIFICATION: 5 μL of 2% NH4H2PO4.

86
9.1.6. Cadmium

9.1.6.1. Graphite furnace atomic absorption for tissue

METHOD: Graphite Furnace Atomic Absorption

DIGEST MATRIX: ~15% 4:1 HNO3:HClO4 by volume.

INSTRUMENT SETTINGS:

Wavelength: 228.9 nm

Lamp: EDL, 5 watts (Perkin-Elmer 0303-60216)

Tube: L’vov, pyro coated

Carrier gas: Argon

Slit width: 0.7

Background correction: Zeeman

Signal mode: Peak area

Scale expansion: None

Read time: 4 sec

Output: To printer

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas

Ramp Hold Flow (mL/min)

Dry 1 80 9 1 300

Dry 2 140 45 5 300

Dry 3 250 20 5 300

Char 800 20 20 300

Atomize 1800 0 4 0

Cleanout 2650 1 3 300

Cool 20 1 10 300

STANDARDS: Addition calibration using 0.96, 2.37, and 4.62 μg/L Cd to SRM

1566a tissue digestate.

TYPICAL SENSITIVITY: Absorbance is approximately 0.100 for 0.02 μg Cd standard.

CALIBRATION: Peak area of standard versus concentration of standard. Compute

the slope, intercept and correlation coefficient using linear, leastsquares regression.

INJECTION VOLUME: 5 μL

MATRIX MODIFICATION: 10 μL of 2% NH4H2PO4.

87
Cadmium

9.1.6.2. Graphite furnace atomic absorption for sediment

METHOD: Graphite Furnace Atomic Absorption

DIGEST MATRIX: ~4% 4:1 HNO3:HClO4 + 12% HF by volume.

INSTRUMENT SETTINGS:

Wavelength: 228.8 nm

Lamp: EDL, 5 watts (Perkin-Elmer 0303-6216)

Tube: L’vov, pyro coated

Carrier gas: Argon

Slit width: 0.7

Background correction: Zeeman

Signal mode: Peak area

Scale expansion: None

Read time: 4 sec

Output: To printer

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas

Ramp Hold Flow (mL/min)

Dry 1 80 9 1 300

Dry 2 140 45 5 300

Dry 3 250 20 5 300

Char 800 20 20 300

Atomize 1800 0 4 0

Cleanout 2650 1 3 300

Cool 20 1 10 300

STANDARDS: Addition calibration using 0.90, 2.22, and 4.35 μg/L Cd to SRM

1646 sediment digestate.

TYPICAL SENSITIVITY: Absorbance is approximately 0.100 for 0.015 μg Cd standard.

CALIBRATION: Peak area of standard versus concentration of standard. Compute

the slope, intercept, and correlation coefficient using linear,

least-squares regression.

INJECTION VOLUME: 5 μL

MATRIX MODIFICATION: 10 μL of 2% NH4H2PO4.

88
9.1.7. Mercury

Cold vapor/gold foil amalgam for tissue or sediment

METHOD: Cold vapor/gold foil amalgam

DIGEST MATRIX: ~25% 5:3.5 HCl and HNO3 by volume for tissues.

~4% 4:1 HNO3 and HClO4 + 12% HF by volume for sediments.

INSTRUMENT SETTINGS:

Reaction volume: 0.020 -1 mL

Diluent: 3% HNO3

Carrier Gas: Ar

Flow rate: 200 mL/min

Scale expansion: Attenuation - mid-range

Integration time: 5 min

Output: Peak integrated by system software

REDUCTANT: 2% SnCl2 in 10% HCl

STANDARDS: 0.5, 1.25, 5, 7.5, and 12.5 μg/L made in 3% HCl using High

Purity standards.

TYPICAL SENSITIVITY: 0.001 μg/g Hg.

CALIBRATION: Instrument read-out is in peak area and input is in ng Hg, so

calibration curve is based on Hg input versus Hg output reading.

The slope, intercept, and correlation coefficient are calculated

using linear, least-squares regression.

INJECTION VOLUME: 1 to 5 mL

REDUCTANT VOLUME: 2.5 mL

MATRIX MODIFICATION: None

89
9.1.8. Lead

Graphite furnace atomic absorption for tissue

METHOD: Graphite Furnace Atomic Absorption

DIGEST MATRIX: ~15% 4:1 HNO3:HClO4 by volume.

INSTRUMENT SETTINGS:

Wavelength: 283.3 nm

Lamp: EDL, 5 ma (Perkin-Elmer 0303-6039)

Tube: L’vov, coated

Carrier gas: Argon

Slit width: 0.7

Background correction: Zeeman

Signal mode: Peak height

Scale expansion: None

Read time: 5 sec

Output: Recorded from Spectrophotometer display

FURNACE PROGRAM:

Step T (°C) Time (sec) Internal Gas

Ramp Hold Flow (mL/min)

Dry 1 80 9 1 300

Dry 2 140 45 5 300

Dry 3 250 20 5 300

Char 800 20 20 300

Atomize 2100 0 4 0

Cleanout 2650 1 3 300

Cool 20 1 10 300

STANDARDS: Method of standard addition calibration at 9.9, 19.6, and 24.4

μg/L Pb to CRM DOLT-1 tissue digestate.

TYPICAL SENSITIVITY: Absorbance is approximately 0.160 for 0.25 μg Pb standard.

CALIBRATION: Peak height of standard versus concentration of standard. Compute

the slope, intercept, and correlation coefficient using linear,

least-squares regression.

INJECTION VOLUME: 5 μL

MATRIX MODIFICATION: 10 μL of 2% NH4H2PO4.

90
9.2. Inductively coupled plasma mass spectrometry

Silver, aluminum, chromium, cadmium, nickel, lead, antimony, and tin in sediments or tissues

METHOD: Inductively coupled plasma mass spectrometry

DIGEST MATRIX: 1 to 10 dilution of digestate with deionized water plus 0.1 mL of 1

ppm In standard.

INSTRUMENT SETTINGS: Instrument settings change on a daily basis as sensitivity is

optimized. This is especially true of lens settings. The following

gives general ranges on parameters that remain relatively

constant in day-to-day operation:

Power: 1250-1500 watts

Coolant flow: 13-14 L/min.

Auxiliary flow: 0.5-1.0 L/min.

Nebulizer flow: 0.85-1.0 L/min.

EM voltage: 2000-3000 volts

Sample uptake: 0.5-1.5 mL/min.

Integration method: Constant area

Integration area: 0.8

Background counts: 35

Dead time: 40 nsec

Quad slew factor: 0.10

PROCEDURE:

Element menu: tissue2

Mass range: 25.98 to 212.05 amu

Number of channels: 2048

Number of sweeps: 100

Dwell time: 320 μsec

Collector type: pulse

Internal standards: In

Skipped mass regions: 28.00 - 43.00, 63.00 - 105.00, 125.00 - 200

STANDARDS: Appropriate calibration standards (representative of sample

concentration) are prepared from dilutions of NIST single- and/or

multi-element standards. Other reference materials (NIST or

second party single/multi-element standards) are used as check

standards. Method of standard addition can be used to produce

instrumental response curve.

TYPICAL SENSITIVITY: Sensitivity is approximately 5 x 104 million counts per sec per

ppm for In115.

CALIBRATION: Constant area integration (using 0.8 of total peak) versus

concentration of standard. Compute the slope, intercept, and

correlation coefficient using linear, least-squares regression.

91
9.3. X-Ray fluorescence

Elements in sediment: Al, As, Cr, Cu, Fe, Mn, Ni, Pb, Si, and Zn.

Elements in tissue: As, Cu, Fe, Mn, Se, Si, and Zn.

METHOD: X-ray fluorescence

Sample preparation: 0.5 g dried and homogenized sediment or tissue pressed in a pellet,

2 cm in diameter.

INSTRUMENT SETTINGS:

Gain: PHA/LTC for Series 80 MCA

Secondary target: Zr

Target gain: 25 eV/channel

Resolution: 182 eV at 6.4 KeV

Timing counter: 12 μsec

Bias: -1000 V

Live time: 1500 sec

Dead time: < 40%

Tungsten tube voltage: 40 V

Tube current: 20 milliamps

92
ORGANIC ANALYSES

93
Extraction and Clean-Up of Sediments for Trace Organic Analysis

Y. Qian, J. L. Sericano, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

Measurement of organic contaminants, such as polychlorinated biphenyls, polynuclear

aromatic hydrocarbons, and chlorinated pesticides, in soil/sediment require the

isolation of these contaminants from the matrices. An aliquot of the homogenized

sediment sample was chemically dried with sodium sulfate and extracted with

dichloromethane using a Soxhlet apparatus. The extract was concentrated and purified

using silica gel/alumina column chromatography before instrumental analysis. Quality

control samples were processed with each batch of samples in a manner identical to the

samples.

1. INTRODUCTION

Assessment of the environmental concentrations of polynuclear aromatic hydrocarbons (PAHs),

chlorinated pesticides and polychlorinated biphenyls (PCBs) requires their measurement in

sediments at trace levels (parts per billion to parts per trillion).

Ten to thirty grams of chemically-dried sediment was Soxhlet extracted with dichloromethane.

The extract was concentrated and purified using silica gel/alumina column chromatography to

remove matrix interferences.

2. SAMPLE COLLECTION, PRESERVATION AND STORAGE

2.1. Sample collection

Sediment was collected and stored in precleaned glass jars and stored frozen (-20 °C).

2.2. Sample preservation and storage

Sediment samples were shipped frozen to the laboratory and stored at -20 °C until analysis.

After subsampling, excess sample was archived at -20 °C in the dark. Extracts were stored in

the dark at or below 4 °C.

3. INTERFERENCES

Method interferences may be caused by contaminants in solvents, reagents, glassware, and

other sample processing hardware, and lead to false positives during instrumental analysis. All

materials used in this method were routinely demonstrated to be free from interferences by

processing procedural blanks identical to samples (one blank per 20 samples or each batch

whichever is more frequent).

Matrix interferences result from co-extraction of compounds other than the analytes of

interest. Elemental sulfur and naturally occurring lipids can cause interferences in the analysis

94
of sediment extracts. Silica gel/alumina cleanup with activated copper was used to remove

interfering materials from the sample prior to analysis.

4. APPARATUS AND MATERIALS

4.1. Labware and apparatus

Glassware was cleaned by washing with Micro cleaning solution and rinsing with water. The

glassware was then combusted in a muffle furnace at 440 °C for at least 4 hr. Solvent rinses

of acetone followed by dichloromethane may be substituted for muffle furnace heating. After

drying and cooling, the glassware was sealed and stored in a clean environment to prevent the

accumulation of dust or other contaminants. Stored glassware was maintained capped with

combusted aluminum foil.

The following labware and equipment is needed to perform the sediment extraction and

purification procedure:

Beaker, 10-mL (for dry weight).

Glass jars, 250-mL or 500-mL glass jars, or other suitable containers

Vials, 1-mL to 7-mL glass vials with Teflon-lined caps

Glass funnels

Flat bottom flasks, 250- and 500-mL

Soxhlet extractor flasks, 40 mm i.d. and condenser

Thimbles, Alundum, medium or coarse, 44-mL round bottom

Concentrator tube, Kuderna-Danish - 25-mL, graduated. Ground glass stoppers are used to

prevent evaporation of extracts

Snyder column, Kuderna-Danish - 3-ball column

Micro reaction vessels, 2.0-mL or 1.0-mL autosampler vials with crimp cap septa

Chromatographic column, 300 mm x 10 mm i.d., with Pyrex glass wool at bottom and

Teflon stopcock

Analytical balance, capable of weighing to 0.0001 mg

Analytical balance, capable of weighing to 0.1 g

Water bath, heated to 60 - 70 °C

Teflon boiling chips, solvent extracted

Syringes, 10 or 25 μL

Disposable glass Pasteur pipettes, 1-mL

Pyrex glass wool, combusted at 400 °C for 4 hr

Nitrogen gas evaporation unit

Volumetric glassware for sample measurement or introduction of internal standards must be

calibrated.

4.2. Reagents

Water (Reagent water that contains no analytes above the method detection limit.)

Sand, combusted at 400 °C for 4 hr

Sodium sulfate (Na2SO4) [7757-82-6], granular, anhydrous, J. T. Baker, ACS Reagent

grade or equivalent combusted at 440 °C for 4 hr and stored at 120 °C prior to use,

cooled to room temperature in a dessicator

Hexane (C6H14) [110-54-3], pesticide quality or equivalent

Dichloromethane (CH2Cl2) [75-09-2], pesticide quality or equivalent

Pentane, Burdick and Jackson pesticide grade or equivalent, lot tested

95
Alumina, Neutral 80-325 MCB chromatographic grade or equivalent Combusted at 400 °C

for 4 hr and stored at 120 °C prior to use

Silica, Grade 923, 100-200 mesh Aldrich 21,447-7 or equivalent stored at 170 °C for 24

hr prior to use

Activated copper turnings, Fisher Scientific, 6575-500, hydrochloric acid washed; water,

methanol, and dichloromethane rinsed.

Activated granular copper, J. T. Baker, analytical grade, 1720-05, 20-30 mesh,

hydrochloric acid washed, water, methanol, and dichloromethane rinsed.

Surrogate spiking solutions

Matrix spike standard

Internal standard solution

5. PROCEDURE

5.1. Sample Preparation

An aliquot of approximately 1 g of sample was weighed in a clean 10-mL beaker. After oven

drying at 63 - 56 °C for at least 24 hr, this aliquot was reweighed and returned to the oven to

further dry the samples for at least another 2 hr before the second weighing. If the difference

between the two weights of the dried aliquot is less than ±0.02 g, the second reading of the

weight was used to calculate the percent solid of the sample. If the difference was greater than

±0.02 g, the oven drying and reweighing was continued until a constant weight was obtained.

A weighed aliquot of sample (10 to 30 g) was chemically dried by mixing with anhydrous

sodium sulfate (30 - 100 g). The mixture of the sample and the sodium sulfate was stirred

continuously with a clean stainless steel spatula until the dried sample was free-flowing. The

sample was transferred to an extraction thimble. The thimble was placed into the Soxhlet

holder. Three hundred mL of dichloromethane, 1 or 2 boiling chips and 2 g of copper turnings

(activated by rinsing with concentrated HCl) were added to the 500-mL flat bottom extraction

flask. The Soxhlet holder with sample thimble inside was attached to the flask and the sample in

the thimble was wetted with approximately 50 mL of CH2Cl2.

After the sediment samples in the thimble were spiked with surrogates [(and matrix spike

solution to the matrix spike (MS) and matrix spike duplicate (MSD)], the samples were

extracted in the Soxhlet apparatus on a hot sand bath for 8 hr. Recycling of the solvent in the

Soxhlet apparatus was maintained at approximately one cycle for every 4 min (i.e., 15 cycles

per hour).

A 3-balled Snyder column was attached to sample extracts in the 500-mL flat bottom flask.

The extracts were concentrated down to 10 - 15 mL on a water bath at 60 - 80 °C. If sediment

or other particulates were present in the sample extract, the extracts were filtered through a

funnel containing glass wool and sodium sulfate. The concentrated sample extract was

transferred to a 25-mL concentrator tube. The flat bottom flask was rinsed two to three times

with dichloromethane and the rinses transferred to the concentrator tube. The sample extract

was then concentrated and solvent changed to about 2 mL of hexane.

5.2. Silica/alumina column cleanup

A glass chromatographic column (30 cm x 10 mm) with a 300-mL reservoir was filled with

dichloromethane. A plug of glass wool and 1 cm of combusted sand were placed in the glass

chromatographic column. Ten grams of alumina (deactivated with 1% water) in CH2Cl2 was

slurry-packed into the column and the alumina allowed to settle. Twenty grams of silica gel

96
(deactivated with 5% water) in CH2

Cl2 was slurry-packed into the column. The silica gel was

allowed to settle. About 1 cm of combusted sand and 1 - 2 cm of activated granular copper was

then placed on top of the packed column. The CH2Cl2 in the column was drained until the solvent

reached the top of the copper. Next 50 mL of pentane were added to the column and the pentane

drained to the top of the copper.

The concentrated sample extract in 2 mL of hexane was transferred to the column using a

disposable pipette. The sample was drained to the top of the copper and the eluent was collected

in a 250-mL flat bottom flask. The concentrator tube that contained the sample extract was

rinsed twice with 1 mL of 50/50 pentane/dichloromethane, and the rinses added to the column.

The solvent was drained to the copper layer and collected in the flask. Two hundred mL of

50/50 pentane/CH2Cl2 was added to the column and collected in the flask at a flow rate of

approximately 1 mL/min. This fraction contained the aromatic and chlorinated hydrocarbons. A

3-balled Snyder column was attached to the 250-mL flat bottom flask and the pentane/CH2Cl2

column fraction and concentrated to 1 mL in hexane on a water bath at 40 - 60 °C.

6. QUALITY CONTROL

Quality control samples were processed in a manner identical to actual samples.

A method blank was run with every 20 samples, or with every sample set, whichever was

more frequent. Blank levels were no more than three times the method detection limit (MDL). If

blank levels for any component were above three times MDL, samples analyzed in that sample

set were re-extracted and reanalyzed. If insufficient sample was available for extraction, the

data was reported and appropriately qualified.

Matrix spike/matrix spike duplicate (MS/MSD) samples were run with every 20 samples, or

with every sample set, whichever was more frequent.

Surrogate standards were spiked into every sample and quality control sample.

Sediment reference material with certified aromatic and chlorinated hydrocarbons

concentrations were analyzed with each sample batch (approximately 20 samples) to

demonstrate the method was in a state of control.

7. CONCLUSIONS

This method has proven to be reliable in quantitatively extracting most organic contaminants

from sediment samples.

97
Extraction of Biological Tissues for Trace Organic Analysis

Y. Qian, J. L. Sericano, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

Measurements of organic contaminants (such as polychlorinated biphenyls, polycyclic

aromatic hydrocarbons and chlorinated pesticides) in biological tissues require the

isolation of these contaminants from the matrices. An aliquot of the homogenized tissue

sample was extracted three times with dichloromethane in the presence of sodium

sulfate by maceration with a tissumizer. An aliquot of 20 mL of the extract was

quantitatively removed for lipid determination. The remaining extract was

concentrated by Kuderna-Danish technique. The concentrated extract was purified using

alumina/silica gel column chromatography. Quality control samples were processed

with each batch of samples in the identical manner as that of the actual samples.

1. INTRODUCTION

Assessment of the environmental levels of chlorinated hydrocarbons [pesticides and

polychlorinated biphenyls (PCBs)] requires their measurement in tissues at trace levels (parts

per billion to parts per trillion).

A tissue sample was homogenized and the sample percent moisture was determined. A

subsample (0.5 - 15 g wet weight) was extracted in the presence of sodium sulfate with

dichloromethane. Prior to concentration, the percent lipids were determined on the extract. The

concentrated extract was then purified using silica gel/alumina column chromatography. Tissue

samples require further purification by geopermeation chromatography (GPC) before

instrumental analysis for pesticides and PCBs.

2. APPARATUS AND MATERIALS

2.1. Apparatus

Balance, top loading with an accuracy of 0.001 g

Balance, Harvard trip dual pan mechanical balance or equivalent

Centrifuge, Beckman Model TJ-6 or equivalent

Electrobalance, Cahn or equivalent, with an accuracy of 0.0001 mg, set on the 250 mg - 1

μg scale

Micropipettes, 50 μL, 100 μL

Nitrogen gas evaporation unit, prefiltered, dried N2

Tissumizer, Pro Scientific, Tek-Mar homogenizer or equivalent

Water bath, heated to 60 - 70 °C

2.2. Labware

Beakers, assorted sizes, borosilicate glass

Boiling chips, Teflon, dichloromethane rinsed

98
Centrifuge bottles, 200-mL, borosilicate glass

Concentrator tubes, Kuderna-Danish, 25-mL, graduated, with ground glass stoppers

Cutting tools (scissors, scalpels, etc.), stainless steel, washed, dried and rinsed with

dichloromethane

Flasks, flat-bottomed, 500-mL capacity, borosilicate glass

Funnels, powder, 85 mm, borosilicate glass

Glass wool, borosilicate glass (combusted)

Pasteur pipettes, 1-mL, disposable, glass

Snyder columns, Kuderna-Danish, 3 ball column

Stoppers, ground glass, 24/40 and 19/22, borosilicate glass

All glassware was washed, and then solvent rinsed or combusted at 440 °C for 4 hr.

2.3. Solvents and reagents

Sodium sulfate (Na2SO4) [7757-82-6], granular, anhydrous, J. T. Baker ACS reagent

grade or equivalent, combusted at 440 °C for 4 hr and stored at 120 °C prior to use,

cooled to room temperature in a dessicator

Hexane (C6H8) Burdick and Jackson pesticide grade or equivalent (each lot tested for

purity)

Methanol (CH3OH), Burdick and Jackson pesticide grade or equivalent

Dichloromethane (CH2Cl2), Burdick and Jackson pesticide grade or equivalent (each lot

tested for purity)

Standards

Matrix spike standard solution, appropriate for pesticide/PCB analysis

Surrogate standard solution, appropriate for pesticide/PCB analysis

Water, Burdick and Jackson HPLC grade or equivalent

3. PROCEDURE

Frozen samples were thawed, either in a refrigerator overnight, or on the counter. After

defrosting, the samples were kept in the refrigerator when not needed.

Careful, thorough mixing of the thawed tissue homogenate was essential since oily or fatty

materials and water tend to migrate to the top of the sample during the freezing/thawing

process.

The top-loading balance was calibrated according to the manufacturer's instructions before

using.

Unless the client has specified the sample to be used for QC samples, the amount of the

homogenized sample was evaluated for the best selection of the QC samples. matrix

spike/matrix spike duplicate (MS/MSD) samples require three times the normal subsample

weight. Duplicates require two times the normal subsample weight.

An aliquot of 0.5 - 15 g (depending on tissue type and availability) was weighed into a 200-mL

centrifuge bottle on the top loading balance. The method blank was prepared for this extraction

procedure contained all solvents, reagents and surrogate spikes and was processed in the same

manner as the samples in the extraction batch. Analytical results for a method blank assess the

presence of contamination introduced during the extraction procedure.

99
An appropriate amount of standard reference material (SRM) was prepared according to the

supplier's instructions. Matrix spike, matrix spike duplicate and duplicate samples were

prepared by weighing sub-aliquot of the tissue sample from the same sample. A laboratory

spike sample (LBS) was prepared by spiking a known volume of a certified spiking standard

solution into a centrifuge bottle. One hundred mL of dichloromethane was added to each

centrifuge bottle containing these samples.

Appropriate amounts of pesticide/PCB surrogate(s) and pesticide/PCB spike solution(s) were

added directly to every sample, blank, SRM or labeled compound spiking solution (LCSS),

matrix spike, matrix spike duplicate, and/or duplicate. The MS/MSD and LBS samples were

processed with each sample batch to determine if the matrix affects analytes recoveries.

Approximately 20 - 50 g of anhydrous sodium sulfate was added to each sample, depending on

the amount of sample used and upon the moisture content of the samples. In general, for small

sample weights (<5 g), 20 - 30 g of sodium sulfate was added. For larger sample weights (5 -

15 g), 50 g of sodium sulfate was added. Similar amounts of sodium sulfate were added to the

method blank.

Tissumizer probes were cleaned with micro-soap and rinsed with tap water. Before using, the

probes were rinsed with water, methanol and dichloromethane in that order. The tissue samples

and QC samples were macerated for 3 min with the tissumizer.

The sample extract was decanted and filtered through sodium sulfate/glass wool funnel. To

filter the samples, the stem of an 85 mm glass powder funnel was lightly packed with glass

wool. Approximately 10 g of sodium sulfate was added to the funnel. The funnel was placed on a

labeled 500-mL flask and the sodium sulfate was pre-wetted with dichloromethane.

The extraction was repeated two more times with 100-mL aliquots of dichloromethane, and the

extracts combined. The sample bottles were rinsed 3 times with small amounts of

dichloromethane after the third extraction step and the rinses poured through the filter into the

500-mL flask.

The total extract volume was marked on the 500-mL flask with a permanent marker. An aliquot

of 20 mL was removed to determine the lipid content.

A few clean boiling chips were added to the 500-mL flat bottom flask and a 3-ball Snyder

column was attached to the flask. The apparatus was placed in a hot water bath (60 - 70 °C)

and the sample was concentrated to about 10 mL.

The apparatus was removed from the water bath and the Snyder column was rinsed with a few

milliliters of dichloromethane into the 500-mL flask. The concentrated extract was

quantitatively transferred to a 25-mL concentrator tube. The 500-mL flat bottom flask was

rinsed at least twice with dichloromethane. The total volume of the extract was determined by

filling the empty 500-mL flasks to the marker with tap water and volume of the water

measured with a 500-mL graduate cylinder.

The sample extract volume in the concentrate tube was further reduced to approximately 2 - 3

mL in a water bath (60 - 70 °C). Then small volumes of hexane were continuously added until

the final volume in the concentrator tube was approximately 2 mL and contained only hexane

(the solution stopped boiling).

100
4. ALUMINA/SILICA GEL CHROMATOGRAPHY

The concentrated extracts in 2 mL of hexane were purified with alumina/silica column

chromatography. The glass columns (30 cm x 1.1 cm i.d.) with 250-mL reservoir and Teflon

stopcock were washed with detergent and rinsed with tap water.

After setting up the columns on the column rack located in a hood, the columns were rinsed

three times with approximately 5 mL methanol followed by three rinses of approximately

5 mL of dichloromethane. The solvent completely covers the inside wall of the column.

A plug of combusted glass wool was inserted into the bottom of the column with a clean glass

rod. The column was filled with approximately 30 mL of dichloromethane. About 2 cm of

combusted sand was added to the column. Deactivation of absorbent was carried out by adding

appropriate amount of water to the activated alumina and/or silica gel and then shaking for at

least 1 hr to thoroughly homogenize the absorbent with water. Ten grams of alumina

deactivated with 1% water was poured into the column while the column was gently tapped.

Twenty grams of silica gel deactivated with 5% water was then slurry packed into each column

on top of the alumina. Approximately 2 cm of anhydrous sodium sulfate was added into the

column on top of the silica gel. The solvent was drained to the top of sodium. The solvent in the

column was changed to pentane by adding about 50 mL of pentane into the column and draining

the pentane to the surface of the sodium sulfate.

The concentrated sample extract was transferred onto the column using a disposable Pasteur

pipette. The solvent was drained to the top of the sodium sulfate and the eluent was collected in

a clean flask. The concentrator tube that contained the sample extract was rinsed with 1 mL of

pentane/dichloromethane (50/50) twice and the rinses transferred to the column. The solvent

was drained and collected in the flask. Approximately 200 mL of pentane/dichloromethane

(50/50) was then added to the column. The sample was collected in the flask at a flow rate of

about 2 mL/min.

After all the solvent passed through the column and was collected in the flask, boiling chips

were added to each flask and a 3-ball Snyder column was attached to the flask. The samples

were then concentrated on a hot water bath (60 - 70°C) to approximately 10 mL. The

concentrated extract was transferred to a concentration tube and the sample further

concentrated to about 0.5 mL.

The concentrated sample was transferred into a tear-shaped (tapered bottom) autosampler

vial. The concentration tube was rinsed with small amounts of dichloromethane twice and the

rinses were transferred into the vial. The final volume in the autosampler vial was less than 1

mL. If more than 1 mL was in the vial, the sample in the vial was concentrated to about 1 mL

using a gentle stream of N2. The sample extract was then ready for further purification using

gel permeation chromatography.

5. QUALITY CONTROL

All quality control samples were processed in a manner identical to actual samples. Quality

control samples included a method blank, a standard reference material, a matrix spike, and a

matrix spike duplicate with every sample set.

6. CONCLUSIONS

This method provides quantitative extraction of most organic contaminants from tissue

samples, including those with high lipid contents.

101
Procedures for the Extraction of Tissues and Purification of Extracts for

Analysis of Polychlorinated Dibenzo- p-dioxins and Polychlorinated

Dibenzofurans

P. Gardinali, L. Chambers, J. L. Sericano, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

Dioxin and furans are compounds of environmental concern because of their high

toxicity at low concentrations. Measurements of these contaminants are made at low

parts per trillion concentration levels. This requires extensive sample clean-up

followed by analysis by high resolution gas chromatography-high resolution mass

spectrometry (HRGC-HRMS).

1. PURPOSE

This procedure describes the protocols for the extraction of biological tissue samples and

purification of the extracts for the measurement of polychlorinated dibenzo-p-dioxins (PCDDs)

and dibenzofurans (PCDFs).

This procedure uses matrix-specific extraction and analyte-specific purification steps to allow

the determination of the 2,3,7,8-substituted PCDD and PCDF isomers using high resolution gas

chromatography/high resolution mass spectrometry. The method provides selective cleanup

procedures to aid in the elimination of interfering contaminants. The method is outlined in

Figure 1.

Samples were spiked with labeled compound spiking solution (LCSS) containing specified

amounts of isotopically 13C12-labeled 2,3,7,8-substituted PCDDs and PCDFs, and homogenized

in a 200-mL centrifuge tube. Tissue samples were dried with 50 g of sodium sulfate (Na2SO4)

and extracted by maceration with a tissumizer three times in 100-mL aliquots of

dichloromethane (CH2Cl2). After extraction, the samples were spiked with a cleanup recovery

standard (CRS), 37Cl4-2,3,7,8-TCDD, to monitor losses through the extract purification steps.

An aliquot for percent lipid determination was then removed. The extract was dried with

sodium sulfate, concentrated and subjected to a bulk purification involving a silica gel/sulfuric

acid slurry. The extract was then processed through three column chromatographic procedures

to remove co-extracted matrix interferences: a mixed bed silica gel column, a basic alumina

column, and an activated charcoal column. All concentration steps were performed using

tetradecane. Due to the high boiling point of tetradecane compared to other solvents, there was

less chances of analyte losses due to volatilization when using this solvent. The final

concentration step reduces the extract to approximately 10 μL tetradecane. An internal

standard solution (ISS) containing selected 13C12-labeled PCDD was added to all final extracts

before analysis bringing the final volume of the extract to 20 μL of tetradecane.

The sample extracts were then ready for identification and determination of the 2,3,7,8-

substituted PCDDs and PCDF isomers.

102
Sample

Homogenization

Extraction

Add 5 μL pf PAR in 1 mL of actone*

Add 10 μL of ISS

Percent lipid

determination

Percent moisture

determination Add 20 μL of LCSS in 1 mL of acetone

Add 5 μL of PAR in 1 mL of acetone*

Acid silica slurry

Mixed bed silica column

Basic alumina column

AX-21 / silica column

Concentrate final eluent to 20 μL C

14

14

Concentrate again to ~20 μL C

HRGC/HRMS analysis

* Precision and recovery spiking solution should be added to MS, MSD, LBS and OPR samples only.

Figure 1. Flow chart for sample processing

103
Interferences in the matrix, solvents, reagents, glassware and other sample processing

hardware may yield discrete artifacts and/or other elevated baselines that cause

misinterpretation of chromatographic data. All materials used during the cleanup procedure

were demonstrated to be free from interferences by analyzing laboratory method blanks at a

frequency of one blank per 20 samples or one with each batch if the number of samples is less

than 20.

The minimum level (ML) for each analyte was defined as the level at which the entire system

must give a recognizable signal and acceptable calibration point. It was equivalent to the

concentration of the lowest calibration standard, assuming that all method specific sample

weights, volumes, and procedures were employed. The MLs vary with degree of chlorination.

Based on 10 g (dry weight) of tissue extracted, the MLs are listed below (refer to EPA method

1613) .

Chlorination (Number of Cl) Concentration (ML)

Tetras (4) 1.0 pg/g

Pentas (5) 5.0 pg/g

Hexas (6) 5.0 pg/g

Heptas (7) 5.0 pg/g

Octas (8) 10.0 pg/g

Minimum Levels were reported for all analytes in all samples.

2. QUALITY CONTROL REQUIREMENTS

2.1. Method blank

A method blank was used to demonstrate that the analytical method was free of contaminating

interferences. The blank was analyzed by executing all of the specified extraction and

purification steps except for the introduction of a sample. The blank was spiked with the labeled

compound spiking solution, the clean-up recovery standard, and the internal standard solution

(ISS) at the appropriate stages of the preparation. A blank was analyzed with each set of 20 or

fewer samples.

2.2. Laboratory blank spike

A laboratory blank spike (LBS) was used to demonstrate analytical precision of the method. It

was prepared by executing all of the specified extraction and extraction purification steps

except for the introduction of a sample. The LBS was spiked with the precision and recovery

spiking solution (PAR), the labeled compound spiking solution (LCSS), the clean-up recovery

standard (CRS) and the internal standard solution (ISS) at the appropriate stages of the

preparation. A LBS was analyzed with each set of 20 or fewer sample.

2.3. Ongoing precision and recovery

An ongoing precision and recovery (OPR) sample was used to demonstrate analytical precision

in the presence of a clean matrix. An OPR was prepared by executing all of the specified

extraction and extract purification steps, using 5 mL of corn oil spiked with the PAR, LCSS,

CRS, and ISS solutions at the appropriate stage of preparation.

104
2.4. Matrix spike

A matrix spike (MS) was used to demonstrate analytical precision in the presence of a

representative matrix. A MS was prepared by executing all of the specified extraction and

purification steps on a selected sample. The MS was spiked with the Precision and Recovery

spiking solution (PAR), the LCSS, CRS, and ISS solutions at the appropriate stages of the

preparation. The MS was analyzed with each set of 20 or fewer samples. A matrix spike

duplicate (MSD) was also analyzed with each set of 20 or fewer samples if required.

2.5. Duplicate

A sample duplicate (DUP) was used to demonstrate matrix homogeneity and analytical precision

in the presence of a representative matrix. A duplicate was prepared by executing all of the

specified extraction and purification steps on replicate portions of a selected sample. The DUP

was spiked with the LCSS, CRS and ISS solutions at the appropriate stages of the preparation.

A duplicate was analyzed with each set of 20 or fewer samples.

2.6. Reference materials

NIST SRM 1944 (for sediment) and NRC CARP-1 (for tissues) were used to demonstrate

analytical accuracy on a certified reference matrix from an independent source. All of the

specified extraction and purification steps were performed on the RM. The RM was spiked with

the LCSS, CRS and ISS solutions at the appropriate stages of the preparation. An RM was

analyzed with each set of 20 or fewer samples. The SRM represented, as closely as possible,

the matrix being analyzed.

2.7. Labeled compound recovery

The percent recovery of the labeled compounds was used to monitor method performance on the

sample matrix. All samples were spiked with LCSS, extracted, purified and analyzed according

to this method.

3. APPARATUS AND MATERIALS

3.1. Glassware and hardware

The following laboratory glassware and hardware was needed to perform the tissue extraction

and purification procedure:

Balance: Top loading with an accuracy of 0.001 g

Chromatography columns: 30 cm x 13 mm (i.d.) with 250-mL reservoir and Teflon

stopcock

Desiccator: Stainless steel construction, no plastic components

Erlenmeyer Flask: 500-mL, with Teflon lined PVC flexible cap

Flat Bottom Flasks: 125-, 250- and 500-mL capacity

Funnels: Glass, 100 mm O.D. wide stem

Glass centrifuge tubes: 200-mL capacity

Graduated cylinders: 10-, 25-, 50-, 100- and 4000-mL capacity

Magnetic stirring plate: Nine-Place, independent stirring speeds for all vessels

Microliter syringes, Micro-pettor pipettes and Disposable micro-capillary pipettes: 1000-,

250-, 100-, 50-, 25-, 20-, 10-, 5-, 4-, 3-, 2- and 1-mL capacity

Microreaction Vessels: 1.0- and 2.0-mL capacity vials with screw cap and Teflon lined

septa

105
Nitrogen blowdown apparatus: Dry heat source and filtered nitrogen stream

Pipettes, disposable, Pasteur: 150 mm x 5 mm (i.d.)

Pipettes, disposable, Serological: 10 mL x 8 mm (i.d.) for preparation of the carbon column

Rotary-evaporator trap: 100 mL

Rotary-evaporator: Buchi, model R144 or equivalent and a low temperature water

recirculator to assure an adequate recovery of the solvents being used.

Stainless steel forceps

Stainless steel knife or shears: for dissecting tissue samples

Tissumizers: Teckmar Polytron homogenizer or equivalent

Vials and Teflon lined caps: 40 mL capacity

Water bath: heated to 60 - 70 °C

No grease, oil, or any other lubricants were used on the desiccator or the rotavap junctions to

avoid contamination.

3.2. Reagents and consumable materials

1 N Sodium hydroxide (NaOH), ACS reagent grade or better from J. T. Baker, Cat. # 3728-

01 or equivalent; prepare a 1 N solution in purified water

Acetone (C3H6O): Burdick and Jackson; Cat. # 010-4; Grade: High Purity Solvent

Cyclohexane (C6H6) [110-82-7], high purity solvent, cat. 053-4. Burdick and Jackson,

Muskegon, MI.

Hexane (C6H14) [110-54-3], capillary GC/GC-MS solvent grade, cat. GC60393-4. Burdick

and Jackson, Muskegon, MI.

Methanol (CH3OH) [67-56-1], high purity solvent, cat. 230-4. Burdick and Jackson,

Muskegon, MI.

Dichloromethane (CCl2H2): Burdick and Jackson; Cat. # 300-4; Grade: High Purity

Purified water: HPLC Grade or better

Sulfuric acid (H2SO4), concentrated J. T. Baker; Cat. # JT9673-00 or equivalent; 95.0-

98.0%; suitable for trace metal analysis.

Tetradecane (C10H22): Fluca Chemical Co.; Cat. # 87140; Highest Grade Available

Toluene (C7H8): Burdick and Jackson; Cat. # 347-4; Grade: High Purity Solvent

Prepurified nitrogen Gas: Nitrogen gas used in final evaporation is purified through an

activated carbon trap. No rubber or plastic is used in the nitrogen delivery system.

Silica gel: Desiccating 8 mesh indication

Anhydrous sodium sulfate (NaSO4): J. T. Baker; Cat. #3891-05 or equivalent; Reagent

Grade: Combusted at 400 °C for 4 hr and stored covered with aluminum foil at 130 °C

Silica gel for column chromatography: E. M. Science; Cat. #7734-5 or equivalent; Silica Gel

60, 70-230 mesh. Activated by heating for more than 16 hr at 170 °C. Stored covered

with aluminum foil at 170 °C.

Basic alumina: E. M. Science; Cat. #AX0612-3 or equivalent; Aluminum Oxide, basic,

chromatographic grade, 80-200 mesh, Alcoa Type GC-20. Combusted at 600 °C for

more than 16 hr. Stored covered with aluminum foil at 130 °C. Used within 3 days,

then reactivated or discarded after that. Recoveries of the targeted analytes were

strongly influenced by the degree of activation of the alumina. Extreme caution must be

taken to avoid use of the adsorbent after 3 days from the original activation.

Activated carbon: AX-21 Carbon (Anderson Development Co.): Wash 100 g of AX-21 carbon

powder (as received) by suspending in 300 mL methanol and subsequently vacuum

filtering through a pre-cleaned glass fiber filter fitted in a 350-mL Buchner funnel

Rinse two times with 100 mL methanol and vacuum dry. Keep the washed AX-21 carbon

at 130 °C for a minimum of 72 hr. Then store in the same oven covered with aluminum

foil at 130 °C.

106
AX-21 carbon/silica gel: Combine 5 g of prepared AX-21 Carbon with 95 g of prepared

Silica Gel in a 500-mL Erlenmeyer with a Teflon lined PVC flexible screw cap (do not

put the cap into the oven). Blend by shaking until a uniform color is achieved. Activate

the mixture at 130 °C for a minimum of 24 hr and store covered with aluminum foil in

the same oven at 130 °C.

H2SO4/silica gel: Prepare by mixing 100 g of concentrated H2SO4 with 150 g of activated

silica gel. Shake and roll on a roller table for a minimum 2 hr. Store in 500-mL

Erlenmeyer flask with Teflon lined PVC flexible screw cap at room temperature.

NaOH/silica gel: Prepare by mixing 33 mL of 1 N NaOH solution and 67 g of activated silica

gel. Store in 500-mL Erlenmeyer flask with Teflon-lined PVC flexible screw cap at

room temperature.

4.2. Analytical standards

Analytical standards were purchased as solutions with certificates of purity, concentration and

authenticity. The LCSS, CRS, PAR and ISS solutions were used as received from the

manufacturer without further treatment. When not being used, standards were stored in the

dark at 4 ± 2 °C in amber glass screw-capped vials with PTFE-lined caps.

4.2.1. Labeled compound spiking solution

The LCSS contained the fifteen 13C12-labeled PCDD and PCDF quantitation standards in nonane at

the nominal concentrations listed in Table 1. Twenty μL of the LCSS were diluted in 1 mL of

acetone and spiked into each tissue sample prior to extraction.

4.2.2. Cleanup recovery standard

The CRS solution contained the cleanup recovery standard contained 40 pg/μL of 37Cl4-

2,3,7,8-TCDD. Five μL of this solution was spiked into each sample extract immediately after

the extraction and before any cleanup procedures were initiated.

4.2.3. Precision and recovery standard

This solution contained the seventeen 2,3,7,8-substituted native PCDD and PCDF isomers at the

nominal concentrations listed on Table 2. Five μL of the precision and recovery standard (PAR)

solution were diluted in 1 mL of acetone and spiked into the selected LBS, MS and MSD prior to

extraction.

107
Table 1. Composition of the labeled compound spiking solution (pg/μL of nonane).

Analyte Concentration (pg/μL)

13C12 - 2,3,7,8-TCDD 100

13C12 -2,3,7,8-TCDF 100

13C12 -1,2,3,7,8-PeCDD 100

13C12 -1,2,3,7,8-PeCDF 100

13C12 -2,3,4,7,8-PeCDF 100

13C12 -1,2,3,4,7,8-HxCDD 100

13C12 -1,2,3,6,7,8-HxCDD 100

13C12 -1,2,3,4,7,8-HxCDF 100

13C12 -1,2,3,6,7,8-HxCDF 100

13C12 -1,2,3,7,8,9-HxCDF 100

13C12 -2,3,4,6,7,8-HxCDF 100

13C12 -1,2,3,4,6,7,8-HpCDD 100

13C12 -1,2,3,4,6,7,8-HpCDF 100

13C12 -1,2,3,4,7,8,9-HpCDF 100

13C12 -OCDD 200

Table 2. Composition of the precision and recovery standard solution (pg/μL of nonane).

Analyte Concentration (pg/μL)

2,3,7,8-TCDD 40

2,3,7,8-TCDF 40

1,2,3,7,8-PeCDD 200

1,2,3,7,8-PeCDF 200

2,3,4,7,8-PeCDF 200

1,2,3,4,7,8-HxCDD 200

1,2,3,6,7,8-HxCDD 200

1,2,3,7,8,9-HxCDD 200

1,2,3,4,7,8-HxCDF 200

1,2,3,6,7,8-HxCDF 200

1,2,3,7,8,9-HxCDF 200

2,3,4,6,7,8-HxCDF 200

1,2,3,4,6,7,8-HpCDD 200

1,2,3,4,6,7,8-HpCDF 200

1,2,3,4,7,8,9-HpCDF 200

OCDD 400

OCDF 400

108
Table 3. Composition of the internal standard solution (pg/μL of nonane).

Analyte Concentration (pg/μL)

13C12 -1,2,3,4-TCDD 200

13C12 -1,2,3,7,8,9-HxCDD 200

4.2.4. Internal standard solution

The ISS contained two 13C12-labeled PCDD isomers at the nominal concentrations listed in Table

3. Ten μL of the ISS were added to the final sample extract before HRGC/HRMS analyses to

determine the percent recoveries for the 13C12-labeled compounds.

4.3. Reference materials

4.3.1. CARP-1

CARP-1 is a ground whole carp reference material for organochlorine compounds which

contains nine of the seventeen target analytes with certified concentrations. This reference

material is available from the National Research Council of Canada.

4.3.2. EDF-2524

EDF-2524 is a clean, natural matrix reference material (fish) gathered in clean waters with a

history of sustaining relatively untainted fish. One analyte, 2,3,7,8-TCDF, is present at a

certified concentration. This reference material is available from Cambridge Isotope

Laboratories.

4.3.3. EDF-2525

EDF-2525 is a contaminated natural matrix reference material (fish) gathered from a polluted

Great Lakes region which contains selected target analytes at certified concentrations within

the calibration range for this method. This reference material is available from Cambridge

Isotope Laboratories.

4.3.4. EDF-2526

EDF-2526 is a fortified natural matrix reference material (fish) which contains all seventeen

target analytes at certified concentrations within the calibration range for this method. This

reference material is available from Cambridge Isotope Laboratories.

4.4. Miscellaneous materials.

Boiling chips: Teflon, solvent rinsed with methane and dichloromethane prior to use.

Glass wool: Combusted at 400 °C for at least 4 hr.

Stirring bars: Teflon coated.

Glass fiber filter paper: Gelman Type A/E or equivalent, Whatmann GF/F or equivalent

109
5. EXTRACTION AND CLEANUP PROCEDURES

5.1. Sample Preparation

An aliquot of the macerated tissue to be extracted was weighed into a centrifuge tube. The

tissue sample size was 10 - 15 g (wet weight), although smaller amounts may be used

depending on the matrix and sample availability.

All glassware was rinsed with methanol, acetone, dichloromethane, toluene, and hexane prior

to use in this extraction procedure.

5.2. Extraction procedure

The tissumizer probe was washed with soap (Micro), rinsed with tap water, and then rinsed

with methanol, acetone, dichloromethane, toluene, and hexane prior to use.

The appropriate amount of tissue was weighed in a solvent-rinsed, labeled 200-mL centrifuge

bottle.

The dioxin LCSS (20 μL) was added to all the samples. The LCSS was first added to 1 mL

acetone in a test tube. This acetone solution was then quantitatively transferred to the

appropriate sample or blank. The test tube was rinsed three times with acetone, and the rinse

solutions were also transferred to the corresponding sample. The PAR standard was added to

the LCSS/acetone mixture which was used for the LBS, MS, and MSD as required. This acetone

solution was then quantitatively transferred to the appropriate sample or blank. The test tube

was rinsed three times with acetone, and the rinse solutions were also transferred to the

corresponding sample. Dichloromethane (100 mL) was added to each sample.

Combusted sodium sulfate (40 mL) was added to each sample immediately before tissumizing.

The tissue was macerated with a tissumizer for three min. A large funnel was placed on top of

a labeled 500-mL flat bottom flask. The stem was plugged with glass wool, and about 2 inches

of sodium sulfate was added. The sodium sulfate was wetted with dichloromethane. The

dichloromethane extract was decanted through the funnel into the labeled 500-mL flat bottom

flask. Another 100 mL of dichloromethane solution was added to the centrifuge bottle and the

extraction step was repeated two more times (a total of three dichloromethane extractions).

5.3. Addition of cleanup recovery standard

The CRS (5 μL) was added to each extract before proceeding to next step.

5.4. Solvent exchange to hexane.

Solvent-cleaned Teflon boiling chips were added to the remaining sample extract in the 500-mL

flask. The Teflon boiling chips were cleaned by rinsing repeatedly with methanol and

dichloromethane (6 - 7 times) and stored in a closed container before use. A three-ball Snyder

column was placed on top of the 500-mL flask. The flask was placed on a hot water bath (60 -

70 °C) and the contents of the flask reduced to about 10 mL. The flask was removed from the

water bath and allowed to cool. The Snyder column was rinsed with hexane. Hexane (120 mL)

was added from a graduated cylinder and the flask was placed back on the bath. The extract

was boiled until the top of the Snyder column became opaque, indicating that the

dichloromethane was gone. After the dichloromethane had completely boiled off, the flask was

removed from the water bath allowed to cool. The Snyder column was rinsed with hexane and

removed. The sample flask was capped with a glass stopper.

110
5.5. Sulfuric acid/silica gel slurry

The H2SO4/silica gel slurry was prepared as described in Section 3.2.1. The labeled flasks

containing the hexane sample extracts were placed on a magnetic stirring plate. A Tefloncoated magnetic stirring bar was added to each flask. Prior to use, the magnetic stirring bars

were rinsed with solvents in the following order: methanol, acetone, dichloromethane, toluene,

and hexane. Metal forceps were rinsed with hexane and used to place the stirring bar in the

sample. The forceps were rinsed with hexane after picking up each stirring bar. The magnetic

stirring plate was turned on and set at medium, making sure the sample did not splash against

the side of the flask. A 50-mL beaker with approximately 40 g of the H2SO4/silica gel slurry

was added to each flask. The samples were stirred for a minimum of 2 hr. The samples were

filtered using a large funnel plugged with combusted glass wool containing combusted sodium

sulfate. The entire contents of the 500-mL flask were poured into the funnel and the flask

rinsed with hexane repeatedly until all the material in the original 500-mL flask was

transferred. After the liquid completely drained through the funnel, the sodium sulfate was

rinsed in the funnel three times with 5 mL of hexane. The 250-mL sample flask was capped with

a glass stopper.

5.6. Rotary evaporation

Fifty μL of tetradecane was added to each sample extract using a 50 μL micropipette. The

rotavap (Rotavapor, Buchi, Brinkman RE121) was set to 250 mbars and the hexane evaporated.

The rotavap trap was rinsed with methanol, acetone, dichloromethane, toluene, and hexane

before the sample flask was attached. The samples were rotavapped to a final volume 50 μL of

tetradecane.

5.7. Mixed bed silica columns

A 300 mm x 13 mm (i.d.) column with a 250-mL reservoir was clamped upside down and rinsed

with methanol, acetone, dichloromethane, toluene, and hexane making sure that the solvents

completely coated and rinsed the inside walls of the column.

The column was turned right-side-up and packed with the following materials added first

(bottom) to last (top):

Combusted glass wool plug

1 scoop of combusted sand (about 1 cm)

1 g activated silica gel

4 g NaOH/silica gel (see Section 3.2.1)

1 g activated silica gel

8 g H2SO4/Silica Gel acid slurry

2 g activated silica gel

1 scoop combusted sodium sulfate (about 1 cm)

A waste jar was placed under the completed column and 25 mL hexane was added as a prerinse. The stopcock was open. The stopcock was closed when the level of hexane was 1 cm

above the sodium sulfate. The bottom tip of the column was rinsed with hexane. The waste jar

was replaced with a 250-mL flat bottom flask. The stopcock was opened and the sample added

to the column using a clean, combusted disposable pipette. The 250-mL flask was rinsed three

times with 2 mL hexane and the rinsate was added to the column using the same pipette for

sample transfer and rinsates. The solvent was drained to the sodium sulfate level. One hundred

and 20 mL of hexane was added to the column with the stopcock open and the hexane eluted at a

111
rate of 1 to 2 drops per second. The column was covered with a piece of aluminum foil. After

the hexane had completely passed through the column, 50 μL tetradecane was added to the

collection flask. The tetradecane was evaporated using a rotavap to a final volume of 50 μL.

5.8. Basic alumina column

A 250 mm x 13 mm (i.d.) column with a 200-mL reservoir was clamped upside down and rinsed

with methanol, acetone, dichloromethane, toluene, and hexane making sure the solvents

completely coated and rinsed the inside walls of the column. The column was turned right-sideup and packed with the following materials added first (bottom) to last (top):

Combusted glass wool

One scoop combusted sand (about 1 cm)

6 g activated alumina (see Section 3.2.1)

1 scoop combusted sodium sulfate (about 1 cm)

A waste jar was placed under the column and the column rinsed with 25 mL of hexane. The

column stopcock was open. The stopcock was closed when the level of the solvent was 1 cm

above the sodium sulfate. The bottom tip of the column was rinsed with hexane. A labeled 125-

mL flat bottom flask was placed underneath the column. With the stopcock closed, the sample

extract was added to the column using a combusted disposable pipette with a glass wool plug.

The flask was rinsed three times with 2 mL hexane and the rinsate added to the column using

the same disposable pipette for the sample and rinsate transfer. The solvent was drained to 1

cm above the surface of the sodium sulfate. Hexane (60 mL) was added to the reservoir and

drained to the sodium sulfate level and the stopcock closed. This fraction was kept until sample

analysis was completed. The waste jar was replaced with a labeled 125-mL flat bottom flask.

A solvent mix (40 mL) of hexane/dichloromethane (70:30) was added to the top of the column.

The 70:30 solvent was made by adding 300 mL dichloromethane to 700 mL hexane in a 1 liter

graduated cylinder. The 70:30 solution was transferred to a 1000-mL flat bottom flask and

mixed thoroughly before each use. The solvent mixture was used for one day and was remade

for each set of samples.

The stopcock was adjusted so the flow was one drip per second and the column was capped with

a piece of solvent rinsed aluminum foil. After the 70:30 solvent completely drained to the

sodium sulfate level, 50 μL of tetradecane was added to the 125-mL sample flask. The solution

was evaporated with a rotavap to a final volume of 50 μL tetradecane.

5.9. Charcoal column

A glass fiber filter paper was placed in a 350-mL Buchner funnel and 100 g of AX-21 carbon

powder was placed on top of the filter paper. Methanol (300 mL) was added to the carbon and

vacuum filtered. The process was repeated two more times with methanol, then vacuum was

used to dry the carbon for 10 sec. The washed carbon was placed in a oven at 130 °C for a

minimum of 72 hr before use.

Activated, room temperature silica gel (95 g) was placed in a 500-mL Erlenmeyer flask.

Washed and cooled AX-21 carbon (5 g) was added to the flask. The flask was capped with a

piece of aluminum foil and shaken by hand until a uniform black color was achieved.

Charcoal columns were made by cutting off both ends of a clean disposable serological pipette.

The pipettes were placed in a 4-L beaker and combusted for 4 hr at 440 °C.

112
The column was packed with combusted glass wool approximately 4 cm from the end. The

column was placed on the rack so the glass wool plug was at the bottom. With a small funnel and

a spatula, 1 cm of activated silica gel was added to the top of the glass wool plug. One gram of

5% AX-21 carbon/silica gel was added being careful not to shake or pack the column. The open

end was plugged with combusted glass wool. The glass wool touched the charcoal but did not

compress it down.

Outline of charcoal column (Section 5.9):

Amount Materials

(TOP) Glass wool plug

1.0 g 5% AX-21 carbon/silica gel

1 cm Activated silica gel

(BOTTOM) Glass wool plug

Twenty-two mL of 50:50 dichloromethane/cyclohexane was added to a labeled 25-mL

concentrator tube. The 50:50 solvent was made by adding equal volume of dichloromethane and

cyclohexane. The 50:50 solvent was used on the day it was prepared and the remaining solvent

was discarded. Fresh solution was prepared for each new set of samples. A waste jar was

placed under the column. The column was pre-rinsed with 5 mL of 50:50 solvent and the

solvent allowed to drain to the glass wool level. The column was turned over and rinsed with

another 5 mL of 50:50 solvent, collecting the solvent in the waste jar. The solvent was drained

to the glass wool level. The combusted disposable pipette with a glass wool plug was used to add

concentrated sample extract from the 125-mL flask (Section 6.10) to the top of the column.

The flask was rinsed with 2 mL of 50:50 solvent three times, using the same pipette for the

removal of the sample and its rinsate, and adding the rinsate to the column. After the solvent

and sample drained to the glass wool level, the remaining 50:50 solvent was added to the

column.

Twenty mL of 75:20:5 solvent mixture was added to the concentrator tubes.

The 75:20:5 solvent mixture was made by mixing 750 mL dichloromethane with 200 mL of

methanol and 50 mL of toluene in a 1000-mL graduated cylinder. This mixture was only good

for one day and was discarded after use. The 20 mL of 75:20:5 was pipetted to the charcoal

column and drained into the waste jar.

When the solvent completely drained down to the glass wool level and stopped dripping, the

column was turned over. A labeled 125-mL flat bottom flask was switched with the waste jar.

The long side (silica gel at the bottom) of the charcoal column was upward. The column was

eluted with 50 mL of toluene. After the toluene completely drained into the 125-mL flask, 20

μL tetradecane was added to the sample extract and rotovapped to a final volume of 20 μL

tetradecane.

5.10. Final evaporation

A 2-mL tear-drop vial was prepared by adding ten (10) μL of tetradecane to each vial. The

samples from the 125-mL flask were transferred to the vials using a disposable, combusted

Pasteur pipette with a glass wool plug. The vials were filled to three quarters of their volume.

New combusted disposable glass Pasteur pipettes were placed in the nitrogen blowdown

apparatus. The Teflon nitrogen hoses were inserted into the pipettes. The 2-mL tear-drop vials

containing the sample were placed on the nitrogen blowdown apparatus and the heat turned on.

The pipette was inserted into the vials, making sure the tip of the pipette did not touch the

sample. The individual controls adjusted until a slight movement of the sample surface was

113
seen. As the volume of the vial was reduced, rinsate from the 125-mL flask was added to the

tear-drop vial. Each 125-mL flask was rinsed three times with about 1 mL of toluene and the

rinsate added to the tear-drop vial. The sample was reduced to a final volume of 20 μL

tetradecane. The vials were removed and placed on a vial tray, leaving the vials uncapped. Ten

μL of ISS was added to each sample using a separate disposable micropipette for each sample.

The vials were replaced on the blowdown apparatus making sure they were in the same position

as before. The sample volumes were reduced to a final volume of 20 μL of tetradecane. The

samples were capped.

6. CONCLUSIONS

This method is an adaptation of EPA regulatory methods that have proven to be reliable

extractions of dioxin and furans at trace (pg/g) concentrations.

114
 Extraction of Sediments for Butyltin Analysis

Y. Qian, J. L. Sericano, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

Measurements of organotin compounds in soil/sediment require the isolation of these

contaminants from the matrices. An aliquot of the freeze-dried sediment sample was

extracted four times with 0.2% tropolone in dichloromethane. The extract was

concentrated by a Kuderna-Danish technique and the solvent exchanged to hexane.

Organotin compounds were hexylated with Grignard reagent and the derivatized extract

purified using silica gel/alumina column chromatography prior to instrumental analysis.

Quality control samples were processed with each batch of samples in a manner

identical to that used for the actual samples.

1. INTRODUCTION

Assessment of the environmental impact of butyltins (i.e., tetra-, tri-, di-, and monobutyltins) requires their measurement in sediments at trace levels (parts per billion to parts

per trillion).

Freeze-dried samples were serially extracted with 0.2% tropolone in dichloromethane. The

extract was then hexylated with a Grignard reagent (see Section 5.3). The hexylated extract

was dried and concentrated. A silica gel/alumina column cleanup step was used prior to

instrumental analysis to remove matrix interferences.

2. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

Sediment samples were collected and placed in pre-cleaned mason jars. Sediment samples were

stored in the dark at or below -20 °C. Sample extracts were stored in the dark at 4 °C.

3. INTERFERENCES

Method interferences may be caused by contaminants in solvents, reagents, glassware, and

other sample processing labware that lead to false positive detections. All materials used in

this method were routinely demonstrated to be free from interferences by processing

procedural blanks using the same procedure as that used for the samples (one blank per 20

samples or each batch whichever was more frequent).

Matrix interferences result from the co-extraction of compounds other than the analytes of

interest. Previous analyses of sediments indicate that matrix interferences were generally

low.

115
4. APPARATUS AND MATERIALS

4.1. Labware and apparatus

Glassware was cleaned by washing with detergent and water and rinsing with water. The

glassware was then combusted in a muffle furnace at 440 °C for at least 4 hr. Solvent rinses

of methanol to dry the glassware followed by dichloromethane may be substituted for the

muffle furnace combustion. After drying and cooling, the glassware was sealed and stored in a

clean environment to prevent the accumulation of dust or other contaminants. Stored glassware

was capped with combusted aluminum foil.

The following glassware was needed to perform the sediment extraction and purification

procedures:

Centrifuge tubes: Corex 50-mL with Teflon-lined screw caps

Chromatographic column: 300 mm x 10 mm i.d., with Pyrex glass wool at bottom and

Teflon stopcock

Concentrator tube: Kuderna-Danish - 25-mL, graduated. Ground glass stoppers are used to

prevent evaporation of extracts.

Disposable glass Pasteur pipettes: 1- and 3 mL

Drying column: 20 mm i.d. Pyrex chromatographic column with glass wool at bottom and

Teflon Stopcock, or Pyrex glass funnel

Evaporative flask: Kuderna-Danish - 250- and 500-mL flat bottom flask

Gas evaporation unit: Nitrogen

Graduated cylinder: 1 or 2 L

Micro reaction vessels: 1.0-mL or 2.0-mL autosampler vials with crimp cap septa

Snyder column: Kuderna-Danish - 3 ball column

Syringes: 10 or 25 μL

Teflon boiling chips: Solvent extracted

Vials: 1-mL to 7-mL glass vials with Teflon-lined caps

Water bath: heated to 60-70 °C

Volumetric glassware for sample measurement or introduction of internal standards was

calibrated.

4.2. Reagents

6 N HCl, 36.5-38%

Alumina oxide: Basic Brockmann 1, standard grade 150 mesh Aldrich 19, 744-3 or

equivalent. Combust 4 hr at 440 °C. Store at 120 °C prior to use.

Hexylmagnesium bromide: 0.5 M in diethyl ether

Internal standard solution

Matrix spike standard

Reagent water: Water containing no analytes above the method detection limit (i.e., HPLC

water).

Silica gel: Grade 923, 100-200 mesh Aldrich 21,447-7 or equivalent. Store at 170 °C

before use.

Sodium sulfate: (ACS) Granular, anhydrous (purified by heating at 440 °C for 4 hr in a

shallow tray, or other suitable method).

Solvents: Methanol (for rinsing), dichloromethane, hexane, pentane (pesticide quality or

equivalent).

Surrogate spiking solutions

Tropolone: Aldrich T8, 970-2, purity 98%

116
5. PROCEDURE

5.1. Sample extraction

An aliquot of about 15 g of freeze dried sediment was weighed into a 50-mL Corex tube.

Appropriate amounts of surrogate standards (approximately 10 times the MDL) were added to

all samples, spikes, and blanks. Appropriate amounts of spiking standards were added to the

matrix spike/matrix spike duplicate (MS/MSD) and/or laboratory blank spike (LBS).

Approximately 30 - 40 mL of 0.2% tropolone in dichloromethane was added to the Corex tube.

The Cortex tube was then sealed with a Teflon screw cap and was shaken on a wrist action

shaker for 1 hr. The sample was centrifuged and the extract decanted into a 500-mL flat

bottom flask. Another 30 mL of 0.2% tropolone in dichloromethane was added into the Cortex

tube and the extraction was repeated three more times.

The sample extract was concentrated in a water bath. A few clean boiling chips were added to

the 500-mL flat bottom flask and a 3-ball Snyder column attached to the flask. The apparatus

was placed in a hot water bath (60 - 70 °C) and the sample concentrated to about 20 mL. The

concentrated extract was transferred to a 50-mL centrifuge tube and further concentrated to 4

- 10 mL. The solvent was exchanged to hexane by adding about 30 mL of hexane to the extract

and continuing the concentration to a final volume of about 10 - 20 mL at which point only

hexane remained.

5.2. Hexylation

Extreme care was taken when handling Grignard reagents because it can react violently with

water. The test tube that contained sample extract was purged with nitrogen and 2 mL of

hexylmagnesium bromide (2 M; Grignard reagent, see below) was added to the sample extract

container. The test tube was then sealed with a Teflon lined screw cap and placed in a 70 °C

water bath for 30 min to hexylate the sample. After cooling, the excessive Grignard reagent

was neutralized with 5 mL of 6 N HCl. The hexylated sample was shaken vigorously before the

organic phase was transferred with a pasture pipette into a 125-mL flat bottom flask. About 15

mL of pentane:CH2Cl2(3/1, v/v) was added to the aqueous phase in the 50-mL centrifuge tube

and the sample shaken vigorously. The organic phase was transferred to the 125-mL flat

bottom flask. The pentane:CH2Cl2extraction was repeated one more time.

The combined hexylated extract in the 125-mL flask was dried by adding 10 - 20 g of

anhydrous Na2SO4 and then concentrated to 1 - 10 mL in a water bath (60 - 70 °C). The

concentrated extract was transferred into a concentrator tube and further concentrated to

about 2 mL.

Grignard reagent was made from magnesium and bromohexane in anhydrous ether. The

apparatus used for making Grignard reagent consisted of a 1-L round bottom flask, a Y-shaped

connector, a condenser, a graduated addition cylinder (250-mL) with a stopcock, and a

desiccating tube filled with desiccant. The condenser and the graduate addition cylinder were

connected to the flask via the Y-shaped connector. The desiccating tube was placed on top of the

condenser so that water vapor could not react with the Grignard reagent being made. All the

glassware was cleaned by rinsing with methanol followed by dichloromethane. After the

glassware was dried in an oven for a few min, it was placed in a dessicator before use.

Approximately 15 g of magnesium turning (98% purity, Aldrich) was weighed into the 1-L

flask. Approximately 100 mL of anhydrous ethyl ether was added immediately to the flask to

117
completely cover the magnesium turnings. The condenser and the addition cylinder were

connected to the flask via the Y-shaped connector. The apparatus was flushed with a stream of

N2 for a few min to remove any water vapor. It was then sealed with a desiccating tube on top

of the condenser and by turning off the stopcock at the bottom of the addition cylinder. It was

critical to maintain a moisture-free environment inside the apparatus because the Grignard

reagent reacts violently with water. A pre-mixed solution of 150 mL of anhydrous ethyl ether

and 90 mL of bromohexane was added to the addition cylinder. The stopcock at the bottom of the

addition cylinder was then opened to let the ether/bromohexane slowly drip into the flask. The

flask was warmed on the water bath (60 - 65 °C) until the solution the flask started to boil.

The apparatus was then removed from the water bath and gently swirled. The process of

heating on the water bath and swirling of the apparatus was repeated until the solution in the

flask started to react with the magnesium turnings continuously without heating, or nearly all

of the bromohexane/ether was in the flask. When the reaction became violent, the flask was

chilled in an ice water bath. When most of the ether/bromohexane had been added to the flask,

the stopcock at the bottom of the addition cylinder was closed. The apparatus was then placed in

an ice water bath for about one hour. The Grignard reagent in the flask was then transferred

into an amber bottle. An aliquot of reagent was tested for its reactivity by testing using with

butyltin standards. Once the satisfactory reactivity of the reagent (as determined by the

recoveries of the derivatized butyltin compounds by GC-FPD) was achieved, the reagent was

used for hexylation of actual samples.

5.3. Silica gel/alumina column cleanup

Glass chromatographic columns (30 cm x 1 cm with 300-mL of reservoir) were filled with 30

mL of pentane. Approximately 10 g of silica gel was poured into the column while the column

was gently tapped to evenly distribute the silica gel. Alternatively, a slurry of silica gel in

pentane may be used to pack the column. About 10 g of alumina was then added to the top of the

silica gel. The solvent in the column was drained to the top surface of the absorbent. The

concentrated hexylated sample extract in 2 mL of hexane was transferred onto the column. The

extract container was rinsed twice with 1 mL of pentane and the rinses were added to the

column. The eluent from the column was collected in a 250-mL flat bottom flask. Another 50 mL

of pentane was added to the column and the sample was eluted at a flow rate of approximately 2

mL/min. The eluent was collected in the 250-mL flat bottom flask. The collected sample

fraction was concentrated to 0.5 to 1 mL on a water bath.

6. QUALITY CONTROL

Quality control samples were processed in a manner identical to actual samples.

A method blank was run with every 20 samples, or with every sample set, whichever was

more frequent. Analyte levels in the Blank were less than three times the method detection

limit (MDL). If blank levels for any component were above three times MDL, the whole sample

set was re-extracted and reanalyzed. If insufficient sample was available for re-extraction,

the data was reported and appropriately qualified.

Matrix spike/matrix spike duplicate (MS/MSD) samples were run with every 20 samples, or

with every sample set, whichever was more frequent. The appropriate spiking level was ten

times the MDL.

Reference materials were extracted with each set of sample and were analyzed when available.

NRC PACS-1 was a sediment reference material certified for butyltins but it is not longer

available. A replacement is currently under preparation.

118
7. CONCLUSIONS

The method of extraction of dried sediment with a shaker and the complexing agent tropolone

followed by hexylation provides reliable extraction and hexylation of tributyltin as well as

other organotins.

119
Extraction of Tissues for Butyltin Analysis

Y. Qian, J. L. Sericano, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

Measurements of organotin compounds in biological tissues require the isolation of

these contaminants from the matrices. An aliquot of the homogenized tissue sample was

extracted three times by maceration with 0.05% tropolone in dichloromethane in the

presence of sodium sulfate. The extract was concentrated by Kuderna-Danish technique

and the solvent was exchanged to 10 mL of hexane. Organotin compounds were

hexylated with Grignard reagent and the derivatized extract was purified using silica

gel/alumina chromatography before instrumental analysis. Quality control samples

were processed with each bath of samples in a manner identical to that of the actual

samples.

1. INTRODUCTION

Assessment of the environmental impact of butyltins, i.e., tetra-, tri-, di-, and monobutyltins,

requires their measurement in tissues at trace levels (parts per billion to parts per trillion).

Tissue samples were serially extracted with 0.05% tropolone in dichloromethane using a

Tissumizer. The extracts were then hexylated with Grignard reagent (see Section 5.3). The

hexylated extract was dried and concentrated. A silica gel/alumina column cleanup step was

used before the instrumental analysis to remove matrix interferences. The extract was then

ready for analysis of butyltins.

2. SAMPLE COLLECTION, PRESERVATION AND STORAGE

Tissue samples were collected and placed in combusted glass jars and were frozen at -20 °C as

soon as possible. The samples were stored at -20 °C in the dark. Sample extracts were stored

in the dark at or below 4 °C.

3. INTERFERENCES

Method interferences may be caused by contaminants in solvents, reagent, glassware, and

other sample processing hardware that lead to false positive detections. All materials used in

this method were routinely demonstrated to be free from interferences by processing

procedural blanks in a manner identical to that used for the samples (one blank per 20 samples

or each batch, whichever was more frequent).

Matrix interferences may be caused by compounds other than the analytes of interest that

were coextracted from the sample. Biogenic materials that cause interferences in the analysis

of tissue extracts were removed prior to GC/FPD analysis by silica gel/alumina

chromatography.

120
4. APPARATUS AND MATERIALS

4.1. Labware and apparatus

Glassware was cleaned by washing with detergent (micro cleaning solution) and rinsing with

water. The glassware was then combusted in a muffle furnace at 440 °C for at least 4 hr.

Solvent rinses of methanol to dry followed by dichloromethane may be substituted for the

muffle furnace heating. After drying and cooling, glassware was sealed and stored in a clean

environment to prevent the accumulation of dust or other contaminants.

The following labware was needed to perform the tissue digestion and purification procedure:

Balance: Top loading with an accuracy of 0.01 g

Boiling chips: Solvent extracted, Teflon

Chromatographic column: 300 mm x 10 mm, with Pyrex glass wool plug at bottom and

Teflon stopcock.

Concentrator tubes: Kuderna-Danish 25-mL, graduated with ground glass stoppers.

Disposable glass Pasteur pipettes: 1- and 3-mL

Electrobalance: Cahn or equivalent, with an accuracy of 0.0001 mg

Erlenmeyer flask: Various sizes

Flat bottom flasks: 125-mL and 500-mL capacity

Gas evaporation unit: Nitrogen

Glass centrifuge bottles: 500-mL capacity

Mason jars

Microliter syringes: 1000-, 500-, 100-, 50- and 10-μL capacity

Microreaction vessels: 2.0-mL or 1.0-mL autosampler vials with crimp cap septa

Pyrex glass wool: Combusted to 400 °C

Snyder column: Kuderna-Danish, 3-ball column

Stainless steel knife or shears: For dissecting animals

Test tubes: Corex 50-mL with Teflon-lined screw caps

Tissumizers: Tekmar; Polytron homogenizer, or equivalent

Vials and Teflon lined caps: 1-mL to 7-mL capacity

Water bath: Heated to 60 - 70 °C

Volumetric glassware used for sample measurement or introduction of internal standards was

calibrated.

4.2. Solvents and reagents

6N HCl

Alumina Oxide: Basic Brockmann 1, standard grade 150 mesh Aldrich 19,744-3 or

equivalent. Combust 4 hr at 440 °C. Store at 120 °C prior to use.

Hexylmagnesium bromide: 2M in diethyl ether

Internal standard solution

Magnesium turnings (98% purity, Aldrich)

Matrix spike standard

Reagent water: Water containing no analytes above the method detection limit (i.e., HPLC

water).

Silica gel: Grade 923, 100-200 mesh Aldrich 21,447-7 or equivalent. Store at 170 °C for

at least 16 hr before use.

Sodium sulfate: (ACS) Granular, anhydrous (purified by heating at 440oC for 4 hr in a

shallow tray or other suitable method).

Solvents: Dichloromethane, hexane, pentane (pesticide quality or equivalent)

121
Surrogate spiking solutions

Tropolone: Aldrich T8, 970-2, purity 98%

5. PROCEDURES

5.1. Preparation of samples

The bivalves were shucked with a stainless steel knife and the tissue was removed from shell.

The tissue was macerated using a Tissumizer or Polytron blender. An aliquot of the macerated

tissue was weighed into a 200-mL centrifuge bottle (2 - 15 g wet weight).

An aliquot of 1 g of macerated tissue was removed and placed in a combusted 10-mL beaker.

This aliquot was dried at 63 - 65 °C. The difference in the weight of the wet sample and the

dried sample was used to determine the percent dry weight.

5.2. Digestion and extraction

Approximately 100 mL 0.05% tropolone in CH2Cl2 and 50 g anhydrous Na2SO4 were added to

the weighed tissue sample (2 - 15 g) in the 200-mL centrifuge bottle. Appropriate amounts of

surrogate standards were added to each sample, and spiking standards to the matrix spike and

matrix spike duplicates. The tissue sample was then macerated for 3 min with the Tissumizer.

The extract was decanted into a 500-mL flat bottom flask (centrifuged at 2000 rpm, if

necessary) through a funnel containing sodium sulfate. The extraction was repeated two more

times with 100 mL of 0.05% tropolone in CH2Cl2 each time.

After the extraction, a 3-ball Snyder column was attached to the 500-mL flat bottom flask. A

few clean boiling chips were added to sample extract. The sample extract was concentrated to

10 - 20 mL on a hot water bath (60 - 70 °C). The sample was then transferred to a 50-mL test

tube. The 500-mL flat bottom flask was rinsed with 5 - 10 mL of hexane and the rinse was

added to the test tube. The sample extract in the test tube was further concentrated on the

water bath and the solvent was exchanged to about 10 mL of hexane by adding hexane while the

dichloromethane was evaporated.

5.3. Hexylation

Extreme care was taken when handling Grignard reagents because it can react violently with

water. After the test tube that contained the sample extract in 10 mL of hexane was purged

with nitrogen, 2 mL of hexylmagnesium bromide (2 M; Grignard reagent) was slowly added into

the sample. The test tube was sealed with a Teflon-lined screw cap and shaken. The hexylation

process occurred while warming the sample in 70 °C in a hot water bath for at least 30 min. At

the end of the 30 min, the samples was taken out of the water bath and allowed to cool to room

temperature. The excess Grignard reagent was neutralized by adding 5 mL of 6 N HCl into the

sample container. The sample was shaken vigorously. The organic phase was transferred with a

pasture pipette into a 125-mL flat bottom flask. The aqueous phase was extracted twice more

with 15 mL of CH2Cl2, and the CH2Cl2 was transferred to the 125-mL flat bottom flask. The

sample was then dried by adding 10 - 20 g sodium sulfate to the 125-mL flask. The solution

was transferred to a concentrator tube and concentrated to about 2 mL.

Grignard reagent was made from magnesium and bromohexane in anhydrous ether. The

apparatus used for making Grignard reagent consists of a 1-L round bottom flask, a Y-shaped

connector, a condenser, a graduated addition cylinder (250-mL) with a stopcock, and a

desiccating tube filled with desiccant. The condenser and the graduate addition cylinder were

122
connected to the flask via the Y-shaped connector. The desiccating tube was placed on top of the

condenser so that water vapor could not react with the Grignard reagent being made. All the

glassware was cleaned by rinsing with methanol followed by dichloromethane. After the

glassware was dried in an oven for a few minutes, it was stored in a dessicator prior to use.

Approximately 15 g of magnesium turnings was weighed into the 1-L flask. Approximately 100

mL of anhydrous ethyl ether was added immediately to the flask to completely cover the

magnesium turnings. The condenser and the addition cylinder were connected to the flask via

the Y-shaped connector. The apparatus was flushed with a stream of N2 for a few minutes to

remove any water vapor. It was then sealed with a desiccating tube on top of the condenser and

by turning off the stopcock at the bottom of the addition cylinder. It was critical to maintain a

moisture-free environment inside the apparatus because the Grignard reagent reacts violently

with water. A pre-mixed solution of 150 mL of anhydrous ethyl ether and 90 mL of

bromohexane was added to the addition cylinder. The stopcock at the bottom of the addition

cylinder was then opened to let the ether/bromohexane slowly drip into the flask. The flask

was warmed on the water bath (60 - 65 °C) until the solution the flask started to boil. The

apparatus was then removed from the water bath and gently swirled. The processing of heating

on the water bath and swirling of the apparatus was repeated until the solution in the flask

started to react with the magnesium turnings continuously without heating, or nearly all of the

bromohexane/ether was in the flask. When the reaction became violent, the flask was chilled in

an ice water bath. When most of the ether/bromohexane was added to the flask, the stopcock at

the bottom of the addition cylinder was closed. The apparatus was then placed in an ice water

bath for about one hour. The Grignard reagent in the flask was then transferred into an amber

bottle. An aliquot of reagent was tested for its reactivity by reacting with butyltin standards.

Once the satisfactory reactivity of the reagent (as determined by the recoveries of the

derivatized butyltin compounds by GC-FPD) was achieved, the reagent was used for hexylation

of actual samples.

5.4. Silica gel/alumina column cleanup

Glass chromatographic columns (30 cm x 10 mm with 300-mL reservoir) were filled with

about 30 mL of pentane. Approximately 10.0 g of silica gel was added into the column. The

column was tapped gently to evenly distribute the silica gel. Alternatively, a slurry of silica

gel in pentane may be used to pack the column. Then 10 g of alumina was added into the column

on the top of the silica gel. The pentane was drained through the column until the surface of the

liquid in the column was just above the top of the alumina.

The hexylated and concentrated sample extract in 2 mL of hexane was transferred onto the

column. The sample was allowed to drain to the top of the alumina. The concentrator tube was

then rinsed twice with 1 mL pentane and the rinses were added to the column. Another 50 mL of

pentane was added to the column and the sample was eluted at a flow rate of approximately 2

mL/min. The eluent was collected in a 250-mL flat bottom flask.

The collected sample fraction was concentrated using a three-ball Snyder column and solvent

was exchanged to 0.5 to 1 mL of hexane on the hot water bath (60 - 70 °C). The samples are

then ready for the butyltin instrumental analyses.

6. QUALITY CONTROL

Quality control samples were processed in a manner identical to actual samples.

123
A method blank was run with every 20 samples, or with every sample set, whichever was

more frequent. The analyte levels in the Blank was required to be less than three times the

method detection limit (MDL). If blank levels for any component were above three times the

MDL, samples analyzed in that sample set were re-extracted and reanalyzed. If insufficient

sample was available for re-extraction, the data was reported and appropriately qualified.

Matrix spike/matrix spike duplicate (MS/MSD) samples were run with every 20 samples, or

with every sample set, whichever was more frequent. The appropriate spiking level was ten

times the MDL.

Reference materials were extracted and analyzed when available.

7. CONCLUSIONS

This tissue extraction was adapted from the standard NS&T organic tissue extraction technique

with the addition of the complexing agent tropolone. The method provides reliable extraction

and hexylation of tributyltin as well as other organotins.

124
Purification of Biological Tissue Samples by Gel Permeation Chromatography

for Organic Analyses

Y. Qian, J. L. Sericano, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

Gel permeation chromatography is an extract purification step which uses a

chromatographic column to separate components in a complex mixture based upon their

molecular size or shape. The size exclusion column used for this procedure separated

lipids and high molecular weight components from target chlorinated and aromatic

hydrocarbons in tissue extracts. An automated gel permeation chromatographic

technique using a high performance liquid chromatography system consisting of an

autosampler, a ultraviolet detector, and an isocratic pump was used to purify sample

extracts. Extracts were processed through a guard column and two size exclusion

columns connected in series and the desired fraction was collected with a fraction

collector. The collected fraction was then concentrated and analyzed for polycyclic

aromatic hydrocarbons, pesticides, and polychlorinated biphenyls using the appropriate

instrumental procedures.

1. INTRODUCTION

Sample extracts, particularly those from biological tissue samples, contain a large amount of

lipids and high molecular weight components. These non-target components may interfere with

the instrumental analyses of polycyclic aromatic hydrocarbons (PAHs), pesticides, and

polychlorinated biphenyls (PCBs). Purification of the sample extracts by alumina/silica gel

column chromatography helps alleviate this problem. However, a large amount of neutral lipids

and high molecular weight components from tissue samples are eluted together with the target

analytes. Further purification of the sample extracts is required in order to analyze the PAHs,

pesticides, and PCBs at parts per billion level. Size exclusion chromatography can separate the

target analytes from the high molecular weight lipids and other components.

2. APPARATUS AND MATERIALS

2.1. Equipment

Autosampler: Thermo Separation Products model AS100 or equivalent.

HPLC isocratic pump: Thermo Separation Products model P1000 or equivalent.

Fraction collector: Isco model Foxy 200 or equivalent.

Size Exclusion column: two connected in series, Phenomenex 250 mm X 22.5 mm

Phenogel 100 Å columns or equivalent.

Phenogel 100 Å guard column: Phenomenex 50 mm x 7.8 mm.

UV absorbance detector: Waters 440 or equivalent, 254 nm.

Hewlett-Packard 3396A integrator

Electrobalance: Cahn or equivalent, with an accuracy of 0.001 mg

Water bath: heated to 60 - 65 °C.

125
2.2. Materials

Boiling chips, Teflon, dichloromethane rinsed.

Concentrator tubes, glass, 25-mL.

Fraction collector vials, 50-mL, 25 x 100 mm.

Autosampler vials, tear-shaped (tapered bottom), 1.2 mL Rainin or equivalent.

Pasteur pipettes: 1-mL, glass, disposable.

Autosampler vials, 2-mL, amber borosilicate glass with Teflon liner caps.

Biphenyl, 99% purity, Aldrich Cat. # B3,465-6 or equivalent.

4,4’-Dibromooctafluorobiphenyl (DBOFB), 99% purity, Aldrich Cat. # 10,199-0 or

equivalent.

Perylene, 99% purity, Aldrich Cat. # P1,129-4 or equivalent.

Dichloromethane, Burdick and Jackson Cat. #300-4, pesticide grade or equivalent, Lot

tested.

Helium, 99.99% purity.

2.3. HPLC calibration standard

Stock solutions

Biphenyl (0.25 mg/mL): 6.25 mg of biphenyl was weighed into a 25-mL volumetric flask

using a Cahn electrobalance. The biphenyl was dissolved in dichloromethane and the solvent

was filled to the 25 mL mark. After the biphenyl was completely dissolved, the solution

was transferred to a clean, labeled bottle with a Teflon-lined cap.

Perylene (0.125 mg/mL): 3.125 mg of perylene was weighed into a 25-mL volumetric

flask and the flask was filled with dichloromethane to the mark. After the perylene was

dissolved, the solution was transferred to a clean, labeled bottle with a Teflon-lined cap.

4,4’-Dibromooctafluorobiphenyl (DBOFB) (0.125 mg/mL): 3.125 mg of DBOFB was weighed

into a 25-mL volumetric flask and the flask was filled to the mark with dichloromethane.

After the DBOFB was dissolved, the solution was transferred to a clean labeled bottle with

a Teflon-lined cap.

2.4. GPC/HPLC calibration standard

The GPC/HPLC calibration standards was prepared by adding 1 mL of each of the stock

solutions to a 250-mL volumetric flask and filling to the mark with dichloromethane.

Concentrations of biphenyl, perylene, and DBOFB should be 1 μg/mL, 0.5 μg/mL, and 0.5

μg/mL, respectively.

When not being used, all solid, stock, and diluted standards were stored in the dark at 20 °C

in the freezer. The solutions were brought to room temperature prior to use. With proper

storage, standard solutions were stable for 12 months.

2.5. GPC/HPLC Calibration

Prior to processing analytical extracts through the GPC/HPLC, the system performance was

checked and the GPC/HPLC was calibrated. The calibration of GPC/HPLC verified the instrument

performance based upon the retention time and area of each of the calibration standards. The

time to start collecting the fractions for actual samples was 1 min before the elution of DBOFB.

For this test, three GPC/HPLC calibration solution and a blank were used to verify the

126
performance criteria. Adjustment and/or re-calibration was performed until the criteria were

met.

The GPC/HPLC calibration standard was brought to room temperature prior to use.

Approximately 1 mL of the GPC/HPLC calibration standard solution was transferred into the

Rainin tapered autosampler vials using a disposable Pasteur pipette. The vials were sealed with

Teflon-lined septa that were pre-rinsed with dichloromethane. The GPC/HPLC calibration

standards were used during instrument calibration and in the sample extract purification

sequence.

Solvent blanks were prepared by filling the autosampler vials with dichloromethane. Blanks

were used during instrument calibration and in the sample extract purification sequence.

The HPLC was set at a flow rate of 7 mL/min. The normal operation pressure of the HPLC

system was from 700 to 900 psi at this flow rate. If the pressure was outside this range, the

source of the pressure variation was identified and corrected.

Three calibration standard vials and one blank vial were placed in the sample tray of the

autosampler. The autosampler was programmed to start the sample sequence from the first

position and inject 1 mL of the sample. The cycle time (the total running time per sample) was

set at 25 min. After placing four collection vials in the fraction collector tray, the sample

sequence was started.

The separation of the three standards was checked on the chromatogram. The three compounds

should be completely separated. DBOFB should elute at approximately 15 min, followed by

biphenyl at about 16 min. Perylene should elute at approximately 18 min. The retention time of

the three peaks should not vary more than 5%, or approximately 0.6 min, among the three

chromatograms. The peak area of DBOFB should be at least 1,000,000 integrator units. The

blank should have no peaks. If the calibration is verified, the system performance is acceptable

and the analysis of samples may begin. If the calibration failed, the GPC/HPLC system must be

checked and the problem must be corrected.

Before starting the sample analysis, the collection time of the fraction collector should be

checked and verified, making sure the collection time corresponds to the elution time of the

calibration standards. The starting time of collection was 1 min before the elution of DBOFB.

The total collection time was 7 min and 30 sec after starting the collection. For example, if the

DBOFB elutes at 15:10 min, the starting collection time is set at 14:10 min and the end of

collection time is set at 21:40 min.

2.6. Preparation of sample extracts for GPC/HPLC

After the extraction of the tissue samples and purification of the extracts using column

chromatography, the sample extracts were concentrated to a final volume of 0.5 mL in

dichloromethane and transferred to tear-drop (tapered bottom) autosampler vials. The final

volume in the autosampler vial should not exceed 1 mL.

For extremely viscous extracts or extracts with high lipid content (greater than 500 mg), the

sample extracts can be diluted and divided into multiple autosampler vials. In this case,

fractions collected from the HPLC are concentrated and combined prior to the addition of the GC

internal standards.

A sample sequence was setup in the autosampler sample tray starting from the first position

using the following sequence: three initial calibration standards, one blank, five samples, one

127
blank, five samples, one blank, one continuing calibration standard (CCS), five samples, one

blank, five samples.

For every five samples an instrument solvent blank was run. For every ten samples an

instrument solvent blank and a CCS was added. Each GPC/HPLC sample sequence must end with

a blank and CCS.

Appropriate number of collection vials was placed in the sample tray of the fraction collector,

following the correct sample sequence in the autosampler tray. The number of the collection

vials should be equal or greater than the number of samples plus the instrument blanks and

calibration standards. The autosampler was programmed to run the sample sequence. The

injection volume was set to inject all of the sample (1 mL) into the column. The cycle time was

set at 25 min. The needle height of the autosampler was adjusted so that the entire sample

extract was injected.

After the sample sequence was completed, the correct number of samples collected by the

fraction collector was verified. The runs of calibration standards were verified to meet the QC

criteria. The instrument blank was verified to contain no peaks. The autosampler vials were

also checked to ensure that the entire extract had been injected. The vials should be empty. If

there is extract remaining in the autosampler vials, the volume of the extract may be brought

up to 1 mL with dichloromethane and reinjected into the HPLC system. The reinjected fraction

collected was then combined with the original collected fraction.

The collected sample vials were placed in a vial rack and 1-2 pieces of boiling chips were

added. The sample was then concentrated to approximately 5-10 mL on the 60-65 °C water

bath. The concentrated sample was transferred to a 25-mL concentrator tube. The collection

vials were rinsed at least three times with dichloromethane and the rinses were added to the

concentrator tube. The transferred sample extract was concentrated and the solvent was

exchanged to 1 mL hexane. After adding appropriate amount of required GC internal standards,

the sample was transferred to a labeled 2-mL brown vial. The vial was capped with a Teflon

lined screw cap and was stored at 0 °C in the dark. The purified sample extract was ready for

instrumental analysis.

3. CONCLUSIONS

This HPLC gel permeation technique allows for the separation of most biological lipids from the

PAHs, pesticides, and PCBs measured. With the lipids removed, reliable quantitations can be

achieved.

128
Quantitative Determination of Polynuclear Aromatic Hydrocarbons by Gas

Chromatography/Mass Spectrometry (GC/MS) - Selected Ion Monitoring (SIM)

Mode

G. J. Denoux, P. Gardinali, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

Polycyclic aromatic hydrocarbons can be determined at low concentrations by mass

spectrometry due to their strong molecular ion response. Therefore, GC/MS in the

selected ion monitoring mode provides unambiguous and sensitive detection for PAHs.

1. INTRODUCTION

The quantitative method described in this document was used to determine polycyclic aromatic

hydrocarbons (PAH) and their alkylated homologues in extracts of water, sediments and

biological tissues. Quantitation was performed by gas chromatography mass spectrometry

(GC/MS) in the selected ion monitoring (SIM) mode. Target analytes are listed in Table 1.

2. APPARATUS AND MATERIALS

The analytical system included a temperature programmable gas chromatograph (HewlettPackard 5890A, or equivalent) equipped with a split-splitless injection port routinely operated

in the splitless mode. A 30-m long x 0.32-mm i.d., 0.25 μm phase thickness DB-5MS fused

silica capillary column was used for the analytical determination. Helium was used as the

carrier gas and the samples were handle by an autosampler capable of making 1 - 4 μL

injections.

The mass spectrometer (HP5970/5972 MSD) was operated at 70 eV electron energy in the

electron impact ionization mode and was tuned to maximize the sensitivity of the instrument

according to the manufacturer’s specifications. The GC capillary column was directly

interfaced with the ion source of the mass spectrometer.

3. REAGENTS

3.1. Surrogate spiking solution

A surrogate spiking solution was made by weighing appropriate aliquots of pure compounds into

a volumetric flask and diluting to volume with dichloromethane [or by using a commercially

available certified standard (NIST or equivalent)]. Surrogates were added to all samples prior

to extraction at a concentration of approximately ten times the MDL.

The compounds in the surrogate solution were naphthalene-d8, acenaphthene-d10,

phenanthrene-d10, chrysene-d12 and perylene-d12. The surrogate solution provides a spike

where approximately 100 μL of solution gave a concentration of 40 ng/mL in the final extract

129
regardless of matrix. All sample analyte concentrations were quantitated based on the

applicable surrogate (Table 1).

3.2. Internal standard solutions

A solution containing each internal standard (fluorene-d10, and benzo[a]pyrene-d12) at 4 μg/mL

was prepared by weighing a certified standard (NIST or equivalent) into a volumetric flask and

diluting to volume with dichloromethane. The GC conditions were set so that the internal

standards were resolved, but elute in close proximity to, the analytes of interest. Sufficient

solution was added to the extract prior to analysis to give a final concentration of the internal

standard of 40 ng/mL.

3.3. Matrix recovery standard spiking solution

A solution containing 2- to 5-ring PAH compounds was used to fortify matrix spike samples

(Table 2). A certified solution (NIST SRM 2260) was purchased and diluted to the appropriate

working concentration. Dibenzothiophene was not present in the SRM and was added to the

solution by weighing the neat material to make a concentration of 1.00 μg/μL. The spiking

solution was used to fortify samples to a final concentration of approximately ten times the

MDL.

3.4. Reference oil solution

A solution of a laboratory reference oil was analyzed as an instrument reference solution with

each analytical batch. The concentration of oil was approximately 0.8 mg/mL. The oil was

weighed into a volumetric and brought to volume with dichloromethane.

4. GC/MS CALIBRATIONS

A five-point calibration curve was established to demonstrate the linear range of the detector.

The recommended standard concentrations used were 20, 100, 250, 500 and 1000 ng/mL.

Standard solutions were made by diluting NIST SRM 1491 to the appropriate concentration.

Dibenzothiophene was not present in the SRM and was added to the calibration solution by

weighing the neat material. When response factors were used, the averaged response factors

for the targeted analytes were kept within a ±15% RSD. Linear regression was used for the

calibration curve evaluation. The control values of the R

2 term were set to be greater than

0.99.

After every 8 - 10 samples, the mass spectrometer response for each PAH relative to the

internal standard was determined using check standards at concentrations of 250 ng/mL. Daily

response factors for each compound were compared to the initial calibration curve. If the

average daily response factors for all analytes were within ±15% of the calibration value,

analyses proceeded. If, for any analyte, the daily response factor exceeded ±25% of

calibration value, a five point calibration was repeated.

Qualitative identification of target compounds was based on relative retention time (RRT)

criteria. Table 3 contains example RRT data for unsubstituted PAHs. RRT windows for alkyl

homologues were based on analysis of the reference oil.

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Table 1. Target compounds.

Compounds IS Surrogate Compounds IS Surrogate

reference reference reference reference

Naphthalene A 1 Benz[a]anthracene B 4

C1-Naphthalenes A 1

C Chrysene B 4

2

-Naphthalenes A 2

C1-Chrysene * B 4 C3

-Naphthalenes A 2

C2-Chrysene * B 4

C4-Naphthalenes * A 2

C3-Chrysene * B 4

C4 Biphenyl A 2 -Chrysene * B 4

Acenaphthylene A 2

Benzo[b]fluoranthene B 4

Acenaphthene A 2 Benzo[k]fluoranthene B 4

Benzo[e]pyrene B 4

Fluorene A 2 Benzo[a]pyrene B 4

C Perylene B 5 1-Fluorenes * A 2

C

Indeno[1,2,3-cd]pyrene B 4

2

-Fluorenes * A 2

Dibenz[a,h]anthracene B 4

C3-Fluorenes * A 2

Benzo[ghi]perylene B 4

Dibenzothiophene A 3

Internal standards

C1-Dibenzothiophenes * A 3

C2-Dibenzothiophenes * A 3 1-Methylnaphthalene A 1

C3-Dibenzothiophenes * A 3 2-Methylnaphthalene A 1

2,6-Dimethylnaphthalene A 2

Phenanthrene A 3 1,6,7-Trimethylnapthalene A 2

C1-Phenanthrenes A 3 1-Methylphenanthrene A 3

C2-Phenanthrenes * A 3

Internal Standards C3

-Phenanthrenes * A 3

C4-Phenanthrenes * A 3

Fluorene-d10 (A)

Benzo[a]pyrene-d12 (B) Anthracene A 3

C1-Anthracenes * A 3

Surrogates C2

-Anthracenes * A 3

C3-Anthracenes * A 3

Naphthalene-d8(1)

C4-Anthracenes * A 3

Acenaphthene-d10 (2)

Phenanthrene-d10 (3) Fluoranthene B 3

Chrysene-d12 (4) C1

-Fluoranthenes * B 3

Perylene-d12 (5)

Pyrene B 3

C1-Pyrene B 3

*Alkylated homologues not included in the calibration solution.

NOTE: Alkylated phenanthrenes and anthracenes, and alkylated fluoranthenes and pyrenes were quantified together as total

alkylated (Cx) phenanthrene/anthracenes and total alkylated (Cx) fluoranthenes/pyrenes. Only the parent compounds and

specific isomers were reported as individual compounds.

131
Table 2. Final concentration of PAH matrix spike solution (in CH2

Cl2).

Compound Spiking solution (μg/mL)

1-Methylnaphthalene 1.31

1-Methylphenanthrene 1.30

2-Methylnaphthalene 1.27

2,6-Dimethylnaphthalene 1.31

2,3,5-Trimethylnaphthalene 1.17

Acenaphthene 1.36

Acenaphthylene 1.26

Anthracene 0.10

Benzo[a]pyrene 1.19

Benzo[b]fluoranthene 1.31

Benzo[e]pyrene 1.31

Benzo[ghi]perylene 1.17

Benzo[k]fluoranthene 1.31

Benz[a]anthracene 1.14

Biphenyl 1.32

Chrysene 1.32

Dibenzothiophene 1.01

Dibenz[a,h]anthracene 0.99

Fluoranthene 1.32

Fluorene 1.01

Indeno[1,2,3-cd]pyrene 1.17

Naphthalene 1.32

Perylene 0.99

Phenanthrene 1.31

Pyrene 1.32

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Table 3. Relative retention times and confidence intervals.*

Absolute 95%

retention time Average Percent confidence

(min) RRT SD RSD Limits

Naphthalene-d8(Surr.) 6:46 1.01 0.02 2.29 0.98-1.04

Naphthalene 6:52 1.01 0.02 2.29 0.97-1.04

2-Methylnaphthalene 8:10 0.87 0.02 2.08 0.84-0.91

1-Methylnaphthalene 8:36 1.20 0.02 2.05 1.16-1.23

Acenaphthylene 10:25 0.96 0.02 1.82 0.93-1.0

Acenaphthene 10:72 0.99 0.02 1.85 0.95-1.02

Fluorene-d10 (IS) 11:50 0.87 0.01 1.73 0.84-0.90

Fluorene 11:96 0.87 0.01 1.74 0.84-0.90

Dibenzothiophene 14:15 0.97 0.02 1.82 0.94-1.01

Phenanthrene 14:35 0.99 0.02 1.59 0.95-1.02

Anthracene 14:45 0.99 0.02 1.60 0.96-1.03

Fluoranthene 17:33 1.13 0.02 1.46 1.10-1.16

Pyrene 17:87 1.16 0.02 1.44 1.12-1.19

Benz[a]anthracene 20:96 0.87 0.01 1.32 0.85-0.90

Chrysene-d12 (Surr.) 20:99 0.90 0.01 1.32 0.87-0.92

Chrysene 21:04 0.88 0.01 1.32 0.85-0.90

Benzofluoranthenes 23:52 0.96 0.01 1.50 0.93-0.99

Benzo[e]pyrene 24:08 0.98 0.02 1.59 0.95-1.02

Benzo[a]pyrene 24:19 0.99 0.02 1.61 0.96-1.02

Perylene 24:38 1.00 0.02 1.84 0.96-1.03

Indeno [1,2, 3-cd] pyrene 26:99 1.112 0.02 2.27 1.06-1.16

Dibenz[a,h]anthracene 27:08 1.11 0.03 2.74 1.05-1.17

Benzo[ghi] perylene 27:71 1.15 0.03 2.42 1.09-1.20

* This table is to serve as an example. Absolute retention times may vary depending on the length and condition of the GC

column.

5. DAILY GC/MS PERFORMANCE TESTS

The mass spectrometer performance was checked daily using perfluorotributylamine (PFTBA)

according to manufacturer’s tuning procedures. These procedures included the optimization of

peak widths, relative abundances, and mass axis calibration for the PFTBA fragments at m/z

69, 219, and 502 according to the recommended criteria. Isotope abundances were also

checked according to the manufacturer’s criteria.

6. GAS CHROMATOGRAPHY/MASS SPECTROMETRY ANALYSES

Just prior to analysis, an aliquot of internal standard solution was added to the sample

producing a final internal standard concentration of approximately 40 ng/mL. Representative

aliquots were injected into the capillary column of the gas chromatograph using the following

conditions:

133
Injector temp: 300 °C

Transfer line temp: 280 °C

Initial oven temp: 40 °C

Initial hold time: 0 min.

Ramp rate: 10 °C

Final temperature: 300 °C

Final hold time: 4 min.

The effluent from the GC capillary column was routed directly into the ion source of the mass

spectrometer (MS). The MS was operated in the selected ion monitoring (SIM) mode using

appropriate windows to include the quantitation and confirmation masses for the PAHs listed in

Table 4. For all compounds detected at a concentration above the MDL the confirmation ion was

checked to confirm its presence.

Compounds identified and quantified below the MDL were appropriately flagged and reported. If

the concentration of any target compound in a sample exceeded the linear range defined by the

standards above, the extract was diluted and reinjected.

7. CALCULATIONS

7.1. Qualitative identification

The extracted ion current profiles of the primary m/z ion for each analyte met the following

criteria:

The retention time fell within ±0.2 min of the retention time of the authentic compound or alkyl

homologue grouping determined by analysis of reference materials.

The alkylated PAH homologue groupings (e.g. C3-naphthalene) appeared as clusters of isomers.

The pattern of each cluster and the retention time window for the cluster was established by

analysis of reference oil solution. The cluster was integrated in its entirety and the total

response was used for quantification.

The relative peak heights of the primary ion compared to the confirmation or secondary ion

masses should be within ±30% of the relative intensities of these masses in a reference mass

spectrum if the compound was correctly identified. The reference mass spectrum was obtained

from reference standards. A compound that does not meet secondary ion confirmation criteria

may indicate interference. The confirmation ion was checked only where interference was

suspected.

7.2. Quantitation

The following formula was used to calculate the response factors of the surrogate relative to

each of the calibration standards.

RF = 

AsCsu

Asu Cs

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Table 4. Quantitations, confirmation ions, and relative abundance.

Analyte Quant. ion Conf. ions Percent abundance*

d8-Naphthalene 136 134 15

Naphthalene 128 127 15

C1-Naphthalenes (including isomers) 142 141 80

C2-Naphthalenes 156 141 ND

C3-Naphthalenes 170 155 ND

C4-Naphthalenes 184 169, 141 ND

d10-Acenaphthene 164 162 95

Acenaphthylene 152 153 15

Biphenyl 154 152 30

Acenaphthene 154 153 98

d10-Fluorene 176 174 85

Fluorene 166 165 95

C1-Fluorenes 180 165 100

C2-Fluorenes 194 179 25

C3-Fluorenes 208 193 ND

d10-Phenanthrene 188 184 ND

Phenanthrene 178 176 20

Anthracene 178 176 20

C1-Phenanthrenes/anthracenes 192 191 60

C2-Phenanthrenes/anthracenes 206 191 ND

C3-Phenanthrenes/anthracenes 220 205 ND

C4-Phenanthrenes/anthracenes 234 219, 191 ND

Dibenzothiophene 184 152, 139 15

C1-Dibenzothiophenes 198 184, 197 25

C2-Dibenzothiophenes 212 197 ND

C3-Dibenzothiophenes 226 211 ND

Fluoranthene 202 101 15

d12-Chrysene 240 236 ND

Pyrene 202 101 15

C1-Fluoranthenes/pyrenes 216 215 60

Benzo[a] anthracene 228 226 20

Chrysene 228 226 30

C1-Chrysenes 242 241 ND

C2-Chrysenes 256 241 ND

C3-Chrysenes 270 255 ND

C4-Chrysenes 284 269, 241 ND

d12-Benz[a]pyrene 264 260 20

Benzo[b]fluoranthene 252 253, 125 30, 10

Benzo[k]fluoranthene 252 253, 125 30, 10

Benzo[e]pyrene 252 253 30, 10

Perylene 264 253 20

d12-Perylene 264 260 ND

Benzo[a]pyrene 252 253, 125 30, 10

Indeno[1,2,3-cd]pyrene 276 277, 138 25, 30

Dibenzo[a,h]anthracene 278 279, 139 25, 20

Benzo[ghi]perylene 276 277, 138 25, 20

ND - Not determined

135
where As

 was the area of the characteristic ion for the parameter to be measured, Asu was the

area of the characteristic ion for the surrogate, Csu was the concentration of the surrogate

(ng/mL), and Cs was the concentration of the target analyte to be measured (ng/mL).

The response factor of alkyl homologues was assumed to be equal to that of respective

unsubstituted parent compounds. Based on these response factors, sample extract

concentrations for each PAH and alkyl homologue grouping was calculated using the following

formula:

Ce = 

AsCsu

Asu RF

where Ce was the sample extract concentration (ng), As was the area of the characteristic

quantitation ion for the target analyte to be measured, Asu was the area of the characteristic

quantitation ion for the surrogate, and Csu was the amount of surrogate added to each extract

(ng).

The actual sample concentration (C) for each compound was calculated using the following

formula:

C = 

Ce

SA

where C was the concentration in sample (ng) and SA was the sample amount (in grams).

8. QUALITY CONTROL/QUALITY ASSURANCE (QA/QC) REQUIREMENTS

8.1. GC/MS tuning

The GC/MS was tuned using PFTBA according to manufacturer’s specifications prior to the

analysis of each QC batch.

8.2. GC/MS initial calibration and continuing calibration checks

A five-point response factor calibration curve was established demonstrating that the analyte

average response factors were within the ±15% RSD criteria (and an R2 of 0.99 or better for

the linear regression) . After every 8 - 10 sample analyses, the mass spectrometer response

factor (RF) for each PAH of interest (Table 1) relative to the internal standard was determined.

The daily response factors for each compound were compared to the initial calibration curve.

The percent difference was calculated using the following equation:

Percent difference = 

(RFI - RFC)

RFI 100

where RFI was the average response factor from initial calibration and RFC was the response

factor from current verification check standard. If the average daily response factors for all

analytes were within ±15% of the calibration value, analyses proceeded. If, for any analyte,

the daily response factor exceeded ±25 percent of the calibration value, a five point calibration

was repeated prior to analysis.

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8.3. Standard reference oil

The standard reference oil was analyzed with all analytical batches. The reference

concentration was defined as the average of all previous analyses plus or minus one standard

deviation. The measured concentration was within ± 25% of the laboratory value on average

for all analytes greater than the MDL and did not exceed ±35% for any individual analyte.

8.4. Method blank analysis

An acceptable method blank did not contain the analytes of interest at concentrations above

three times greater than the MDL. If the method blank exceeds this criteria, the analytical

procedure was out of control and the source of the contamination was investigated and

corrective measures were taken and documented before further sample analysis proceeded. The

first corrective action was to re-inject the blank to confirm the out-of-control event. If the

blank still exceeded the criteria, all samples in that batch were re-extracted and re-analyzed.

8.5. Surrogate compound analysis

All samples, including quality control samples, were spiked with deuterated PAH surrogate

compounds. The surrogate compounds were spiked into the sample prior to extraction to

determine sample matrix effects associated with sample preparation and analysis. Surrogates

included naphthalene-d8, acenaphthene-d10, phenanthrene-d10, chrysene-d12 and perylene-d12.

Just prior to analysis, the extract was spiked with a solution containing the GC Internal

Standards. These compounds were fluorene-d10 and benzo[a]pyrene-d12. The recovery of the

surrogates were monitored in each sample using the relative response factor of the Surrogate

to the Internal Standard.

Percent SUR recovery = 

ASUR C IS

CSUR AIS RFSUR

where AIS was the area of the characteristic ion for the appropriate internal standard, ASUR

was the area of the characteristic ion for the surrogate, CSUR was the amount in ng of

deuterated surrogate added to the sample, CIS was the amount in ng of deuterated internal

standard added to the sample extract and RFSUR was the response factor for the surrogate.

The laboratory took corrective action whenever the recovery for any surrogates, except

perylene-d12, was less than 40% or greater than 120%.

The following corrective actions were taken:

a. The calculations were checked to assure there were no errors.

b. The internal standard and surrogate solutions were checked for degradation,

contamination, etc., and the instrument performance was checked.

c. If the surrogate recovery was outside the control limits, the secondary ion was used to

check the quantitation of the surrogate. If the secondary ion was within the control limits,

this recovery was appropriately annotated.

d. If the upper control limit was exceeded for only one surrogate, and the instrument

calibration and surrogate standard concentration were in control, it was concluded that an

interference specific to the surrogate was present that resulted in high recovery and that

137
this interference did not affect the quantitation of other target compounds. The presence of

this type of interference was confirmed by evaluation of chromatographic peak shapes. To

correct for the underestimation of the analyte concentration based on this surrogate, the

analytes were quantitated using an alternate surrogate that was chromatographically

closest to the surrogate exhibiting interference.

e. If the surrogate could not be measured because the amount and nature of the

hydrocarbon in the sample, the analytes based on that surrogate will be quantitated based

on the closest surrogate. The surrogate recovery was appropriately qualified.

f. If the native concentration of hydrocarbons were high and required that a dilution for

quantitation purposes be made, a known aliquot of the extract was sampled and diluted. One

hundred (100) μL of surrogate and 100 μL of internal standard were added and the volume

brought to 1.0 mL. The appropriate dilution factor was used in the quantitation software for

the sample and surrogate recoveries were assumed to be 100% for the re-analyses. The

recovery in these cases and the surrogate recoveries were not reported but qualified with

a “D” to denote the dilution.

g. If the steps above failed to reveal a problem, the sample or extract was then reextracted. If reanalysis of the new extract yielded surrogate recoveries within the stated

limits, then the reanalysis data was reported. If reanalysis did not yield acceptable

recoveries, the data was listed as out of control because of matrix effects.

8.6. Matrix spike analysis

The laboratory analyzed a matrix spike and matrix spike duplicate (MS/MSD) with every 20

samples or with every sample set, whichever was more frequent. A sample randomly chosen

was split into three subsamples and two subsamples were fortified with the matrix spiking

solution containing the compounds listed in Table 2. The acceptable matrix spike recovery

criteria was a recovery for all 25 compounds between 50 and 120% for at least 80% of the

analytes.

If the matrix spike criteria were not met, the matrix spike analysis was repeated. If the

subsequent matrix spike analysis met the criteria, then the reanalysis data was reported. If the

matrix spike criteria were not met on re-injection, the sample set was re-extracted.

8.7. Standard Reference Material

When available, a standard reference material was extracted and analyzed with each batch of

samples. Target concentrations were defined as the range of the certified value plus or minus

the 95% confidence intervals found in the SRM certification. The measured concentration was

within ±30% of the target concentration on average for all analytes either certified or noncertified with concentrations greater than 10 times the MDL.

8.8. Method detection limit

The actual analytical method detection limit (MDL) was determined following procedures

outlined in Federal Register (1984), Vol. 49, No. 209: 198-199.

138
9. CONCLUSIONS

The PAH quantitation method is very rigorous because PAHs have very strong molecular ion

peaks under the mass spectrometric conditions used. Also the availability of labeled surrogates

internal standards of many of the analytes makes very accurate determinations of analyte

concentrations possible.

139
Quantitative Determination of Tetra- Through Octa-Polychlorinated Dibenzo- pdioxins and Dibenzofurans by Isotope Dilution High Resolution Gas

Chromatography/High Resolution Mass Spectrometry

L. Chambers, P. Gardinali, J. L. Sericano, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

This method was used for the determination of tetra- through octa-chlorinated dibenzop-dioxins (PCDDs) and dibenzofurans (PCDFs) in sediment and tissue samples by high

resolution gas chromatography/high resolution mass spectrometry. The seventeen

2,3,7,8-substituted PCDDs/PCDFs were determined by this method at pg/g levels. The

minimum levels reported for the target compounds are the levels at which the

PCDDs/PCDFs can be determined with no interferences present. The procedures

described here were largely based on the protocols described in EPA Method 1613 and

EPA Method 8290 and designed to meet or exceed the quality control criteria outlined in

those methods. The analyst is permitted to modify the method provided that all

performance criteria in this method are met.

1. PURPOSE AND SUMMARY

This procedure was used to provide quantitative determination of tetra- through octapolychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in sample extracts using

isotope dilution high resolution gas chromatography/high resolution mass spectrometry

(HRGC/HRMS).

The instrumental protocols described in this procedure were applied to the quantitative analysis

of extracts from sediment and bivalve tissues.

The chlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) determined by this

method and their Chemical Abstract Service (CAS) Registry numbers are listed in Table 1. The

retention time references, quantitation references, relative retention times and minimum

levels for determination of PCDDs and PCDFs using this method are listed in Table 2. The

minimum level (ML) for each analyte was defined as the level at which the entire system must

give a recognizable signal and an acceptable calibration point. It was equivalent to the

concentration of the lowest calibration standard, assuming that all method specific sample

weights, volumes, and procedures were employed. The ML varied with the degree of

chlorination. The sample-specific estimated detection limit (EDL) was the concentration of an

analyte required to produce a signal with a peak height at least three times the background

signal level. An EDL was calculated for each 2,3,7,8-substituted isomer that was not detected.

The quantitation software (OPUSQuan) supplied by the instrument manufacturer calculated a

sample specific-EDL using an algorithm based on the criteria outlined in EPA Method 8290,

Section 7.9.5.1.1. The analyte concentrations of the calibration standards (CS1-CS5) used for

instrument calibration are listed in Table 3.

140
Table 1. Chlorinated dibenzo-p-dioxins and dibenzofurans determined by isotope dilution high

resolution gas Chromatography (HRGC)/high resolution mass spectrometry (HRMS).

CDDs/CDFs* CAS Registry Labeled analog CAS Registry

2,3,7,8-TCDD 1746-01-6 13C12-2,3,7,8-TCDD 76523-40-5

37Cl4

-2,3,7,8-TCDD 85508-50-5

Total TCDD 41903-57-5 ---- ----

2,3,7,8-TCDF 51207-31-9 13C12-2,3,7,8-TCDF 89059-46-1

Total-TCDF 55722-27-5 ---- ----

1,2,3,7,8-PeCDD 40321-76-4 13C12-1,2,3,7,8-PeCDD 109719-79-1

Total-PeCDD 36088-22-9 ---- ----

1,2,3,7,8-PeCDF 57117-41-6 13C12-1,2,3,7,8-PeCDF 109719-77-9

2,3,4,7,8-PeCDF 57117-31-4 13C12-2,3,4,7,8-PeCDF 116843-02-8

Total-PeCDF 30402-15-4 ---- ----

1,2,3,4,7,8-HxCDD 39227-28-6 13C12-1,2,3,4,7,8-HxCDD 109719-80-4

1,2,3,6,7,8-HxCDD 57653-85-7 13C12-1,2,3,6,7,8-HxCDD 109719-81-5

1,2,3,7,8,9-HxCDD 19408-74-3 13C12-1,2,3,7,8,9-HxCDD 109719-82-6

Total HxCDD 34465-46-8 ---- ----

1,2,3,4,7,8-HxCDF 70648-26-9 13C12-1,2,3,4,7,8-HxCDF 114423-98-2

1,2,3,6,7,8-HxCDF 57117-44-9 13C12-1,2,3,6,7,8-HxCDF 116843-03-9

1,2,3,7,8,9-HxCDF 72918-21-9 13C12-1,2,3,7,8,9-HxCDF 116843-04-0

2,3,4,6,7,8-HxCDF 60851-34-5 13C12-2,3,4,6,7,8-HxCDF 116843-05-1

Total-HxCDF 55684-94-1 ---- ----

1,2,3,4,6,7,8-HpCDD 35822-46-9 13C12-1,2,3,4,6,7,8-HpCDD 109719-83-7

Total-HpCDD 37871-00-4 ---- ----

1,2,3,4,6,7,8-HpCDF 67562-39-4 13C12-1,2,3,4,6,7,8-HpCDF 109719-84-8

1,2,3,4,7,8,9-HpCDF 55673-89-7 13C12-1,2,3,4,7,8,9-HpCDF 109719-94-0

Total-HpCDF 38998-75-3 ---- ----

OCDD 3268-87-9 13C12-OCDD 114423-97-1

OCDF 39001-02-0 not used

* Chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans

TCDD = Tetrachlorodibenzo-p-dioxin TCDF = Tetrachlorodibenzofuan

PeCDD = Pentachlorodibenzo-p-dioxin PeCDF = pentachlorodibenzofuran

HxCDD = Hexachlorodibenzo-p-dioxin HxCDF = Hexachlorodibenzofuran

HpCDD = Heptachlorodibenzo-p-dioxin HpCDF = Heptachlorodibenzofuran

OCDD = Octachlorodibenzo-p-dioxin OCDF = Octachlorodibenzofuran

141
Table 2. Retention time references, quantitation references, relative retention times, and

minimum levels for CDDs and CDFs.

PCDD/PCDF Retention time and Minimum

.....................quantitation reference....................... .....................level*...................

Water Solid Extract

(pg/L) (pg/g) (pg/μL)

Compounds using 13C12-1-2,3,4-TCDD as the injection internal standard

2,3,7,8-TCDF 13C12-2,3,7,8-TCDF 0.999-1.003 10 1 0.5

2,3,7,8-TCDD 13C12-2,3,7,8-TCDD 0.999-1.002 10 1 0.5

1,2,3,7,8-PeCDF 13C12-1,2,3,7,8-PeCDF 0.999-1.002 50 5 2.5

2,3,4,7,8-PeCDF 13C12-2,3,4,7,8-PeCDF 0.999-1.002 50 5 2.5

1,2,3,7,8-PeCDD 13C12-1,2,3,7,8-PeCDD 0.999-1.002 50 5 2.5

13C12-2,3,7,8-TCDF 13C12-1,2,3,4-TCDD 0.923-1.103 NA NA NA

13C12-2,3,7,8-TCDD 13C12-1,2,3,4-TCDD 0.976-1.043 NA NA NA

37Cl4

-2,3,7,8-TCDD 13C12-1,2,3,4-TCDD 0.989-1.052 NA NA NA

13C12-1,2,3,7,8-PeCDF 13C12-1,2,3,4-TCDD 1.000-1.425 NA NA NA

13C12-2,3,4,7,8-PeCDF 13C12-1,2,3,4-TCDD 1.011-1.526 NA NA NA

13C12-1,2,3,7,8-PeCDD 13C12-1,2,3,4-TCDD 1.000-1.567 NA NA NA

Compounds using 13C12-1,2,3,7,8,9-HxCDD as the injection internal standard

1,2,3,4,7,8-HxCDF 13C12-1,2,3,4,7,8-HxCDF 0.999-1.001 50 5 2.5

1,2,3,6,7,8-HxCDF 13C12-1,2,3,6,7,8-HxCDF 0.997-1.005 50 5 2.5

1,2,3,7,8,9-HxCDF 13C12-1,2,3,7,8,9-HxCDF 0.999-1.001 50 5 2.5

2,3,4,6,7,8-HxCDF 13C12-2,3,4,6,7,8-HxCDF 0.999-1.001 50 5 2.5

1,2,3,4,7,8-HxCDD 13C12-1,2,3,4,7,8-HxCDD 0.999-1.001 50 5 2.5

1,2,3,6,7,8-HxCDD 13C12-1,2,3,6,7,8-HxCDD 0.998-1.004 50 5 2.5

1,2,3,7,8,9-HxCDD 1.000-1.019 50 5 2.5

1,2,3,4,6,7,8-HpCDF 13C12-1,2,3,4,6,7,8-HpCDF 0.999-1.001 50 5 2.5

1,2,3,4,7,8,9-HpCDF 13C12-1,2,3,4,7,8,9-HpCDF 0.999-1.001 50 5 2.5

1,2,3,4,6,7,8-HpCDF 13C12-1,2,3,4,6,7,8-HpCDD 0.999-1.001 50 5 2.5

OCDF 13C12-OCDD 0.999-1.008 100 10 5.0

OCDD 13C12-OCDD 0.999-1.001 100 10 5.0

13C12-1,2,3,4,7,8-HxCDF 13C12-1,2,3,7,8,9-HxCDD 0.944-0.970 NA NA NA

13C12-1,2,3,6,7,8-HxCDF 13C12-1,2,3,7,8,9-HxCDD 0.949-0.975 NA NA NA

13C12-1,2,3,7,8,9-HxCDF 13C12-1,2,3,7,8,9-HxCDD 0.977-1.047 NA NA NA

13C12-2,3,4,6,7,8-HxCDF 13C12-1,2,3,7,8,9-HxCDD 0.959-1.021 NA NA NA

13C12-1,2,3,4,7,8-HxCDD 13C12-1,2,3,7,8,9-HxCDD 0.977-1.000 NA NA NA

13C12-1,2,3,6,7,8-HxCDD 13C12-1,2,3,7,8,9-HxCDD 0.981-1.003 NA NA NA

13C12-1,2,3,4,6,7,8-HpCDF 13C12-1,2,3,7,8,9-HxCDD 1.043-1.085 NA NA NA

13C12-1,2,3,4,7,8,9-HpCDF 13C12-1,2,3,7,8,9-HxCDD 1.057-1.151 NA NA NA

13C12-1,2,3,4,6,7,8-HpCDD 13C12-1,2,3,7,8,9-HxCDD 1.086-1.110 NA NA NA

13C12-OCDD 13C12-1,2,3,7,8,9-HxCDD 1.032-1.311 NA NA NA

Relative retention time criteria apply to reporting for EPA Method 1613. Unless EPA Method 1613 is specifically required, the -1 to + 3 sec RT

criteria are used (Section 3.2.1.1).

*

The Minimum Level (ML) for each analyte is defined as the level at which the entire analytical system must give a recognizable signal and acceptable

calibration point. It is equivalent to the concentration of the lowest calibration standard, assuming that all method-specific sample weights, volumes,

and cleanup procedures have been employed.

The retention time reference for 1,2,3,7,8,9-HxCDD is 13C12-1,2,3,6,7,8-HxCDD, and 1,2,3,7,8,9-HxCDD is quantified using the averaged

responses for 13C12-1,2,3,4,7,8-HxCDD and 13C12-1,2,3,6,7,8-HxCDD.

142
Table 3. Concentration of PCDDs and PCDFs in calibration (CS) and calibration verification (VER

CS) solutions.

CDD/CDF CS1 CS2 VER CS3 CS4 CS5

(pg/μL) (pg/μL) (pg/μL) (pg/μL) (pg/μL)

2,3,7,8-TCDD 0.5 2 10 40 200

2,3,7,8-TCDF 0.5 2 10 40 200

1,2,3,7,8-PeCDD 2.5 10 50 200 1000

1,2,3,7,8-PeCDF 2.5 10 50 200 1000

2,3,4,7,8-PeCDF 2.5 10 50 200 1000

1,2,3,4,7,8-HxCDD 2.5 10 50 200 1000

1,2,3,6,7,8-HxCDD 2.5 10 50 200 1000

1,2,3,7,8,9-HxCDD 2.5 10 50 200 1000

1,2,3,4,7,8-HxCDF 2.5 10 50 200 1000

1,2,3,6,7,8-HxCDF 2.5 10 50 200 1000

1,2,3,7,8,9-HxCDF 2.5 10 50 200 1000

2,3,4,6,7,8-HxCDF 2.5 10 50 200 1000

1,2,3,4,6,7,8-HpCDD 2.5 10 50 200 1000

1,2,3,4,6,7,8-HpCDF 2.5 10 50 200 1000

1,2,3,4,7,8,9-HpCDF 2.5 10 50 200 1000

OCDD 5.0 20 100 400 2000

OCDF 5.0 20 100 400 2000

13C12-2,3,7,8-TCDD 100 100 100 100 100

13C12-2,3,7,8-TCDF 100 100 100 100 100

13C12-1,2,3,7,8-PeCDD 100 100 100 100 100

13C12-1,2,3,7,8-PeCDF 100 100 100 100 100

13C12-2,3,4,7,8-PeCDF 100 100 100 100 100

13C12-1,2,3,4,7,8-HxCDD 100 100 100 100 100

13C12-1,2,3,6,7,8-HxCDD 100 100 100 100 100

13C12-1,2,3,4,7,8-HxCDF 100 100 100 100 100

13C12-1,2,3,6,7,8-HxCDF 100 100 100 100 100

13C12-1,2,3,7,8,9-HxCDF 100 100 100 100 100

13C12-1,2,3,4,6,7,8-HpCDD 100 100 100 100 100

13C12-1,2,3,4,6,7,8-HpCDF 100 100 100 100 100

13C12-1,2,3,4,7,8,9-HpCDF 100 100 100 100 100

13C12-OCDD 200 200 200 200 200

Cleanup standard

37Cl4

-2,3,7,8-TCDD 0.5 2 10 40 200

Internal standards

13C12-1,2,3,4-TCDD 100 100 100 100 100

13C12-1,2,3,7,8,9-HxCDD 100 100 100 100 100

143
2. QUALITY CONTROL

2.1. Instrument criteria

2.1.1. Mass spectrometer performance

The mass spectrometer was operated in the electron ionization mode. A static resolving power

of at least 10,000 (10% valley definition) was demonstrated at appropriate masses before any

analyses were performed. Static resolving power checks were performed at the beginning of

each 12 hr period of operation. Corrective action was implemented whenever the resolving

power does not meet the requirement.

Analysis time for PCDDs and PCDFs exceed the long term mass stability of the mass

spectrometer. Because the instrument was operated in high-resolution mode, mass drifts of a

few parts per million can have serious adverse effects on instrument performance. Therefore,

a mass drift correction was mandatory. A lock-mass ion was selected from the reference

compound perfluorokerosene (PFK) used for tuning the mass spectrometer. The selection of the

lock-mass ion was dependent on the masses of the ions monitored within each descriptor (see

Table 4). Any acceptable lock-mass ion at any mass between the lightest and heaviest ion in

each descriptor can be used to monitor and correct mass drifts.

The level of the reference compound (PFK) metered into the ion chamber during HRGC/HRMS

analyses was adjusted so that the amplitude of the most intense selected lock-mass ion signal

did not exceed 10% of the full scale deflection for a given set of detector parameters.

Suggested lock-mass ions are listed in Table 4.

Documentation of the instrument resolving power was accomplished by recording the peak

profile of the reference compound peaks within each descriptor. The format of the peak profile

representation allowed manual determination of the resolution, i.e., the horizontal axis was a

calibrated mass scale. The result of the peak width measurement performed at the 10% valley

definition did not exceed 100 ppm for all reference compound peaks recorded.

Total cycle time (dwell times plus switching times) for all ions within a single descriptor were

one second or less.

2.1.2. GC column performance

Using the GC column performance check solution (CPSM/WDM, see Table 5), the

chromatographic separation between 2,3,7,8-TCDD and the peaks representing any other

unlabeled TCDD isomers were resolved with a valley of <25%, where:

Eq. 1 Valley percent = 

x

y

 100

x is height of the valley between 2,3,7,8-TCDD and the closest eluting isomer, and y is the

peak height of 2,3,7,8-TCDD.

When confirmation analysis of the 2,3,7,8-TCDF isomer was required, chromatographic

separation between 2,3,7,8-TCDF and the peaks representing any other unlabeled TCDF isomers

was resolved with a valley of <25 percent using Eq. 1, replacing TCDD with TCDF.

144
Table 4. Descriptors, exact m/z's, m/z types, and elemental compositions of the CDDs and

CDFs.

Descriptor Exact m/z* m/z type Elemental composition Substance

1 292.9825 Lock C7 F11 PFK

303.9016 M C12 H4

35Cl4O TCDF

305.8987 M+2 C12 H4

35Cl337ClO TCDF

315.9419 M 13C12H4

35Cl4O TCDF

317.9389 M+2 13C12H4

35Cl437Cl3O TCDF

319.8965 M C12H4

35Cl4O2

TCDD

321.8936 M+2 C12H4

35Cl3O2

TCDD

327.8847 M C12H4

37Cl4O2

TCDD

330.9792 QC C7F13 PFK

331.9368 M 13C12H4

35Cl4O2

TCDD

333.9339 M+2 13C12H4

35Cl337ClO2

TCDD

375.8364 M+2 C12H4

35Cl537ClO HxCDPE

2 339.8597 M+2 C12H3

35Cl437ClO PeCDF

341.8567 M+4 C12H3

35Cl337Cl2O PeCDF

351.9000 M+2 13C12H3

35Cl437ClO PeCDF

353.8970 M+4 13C12H3

35Cl337Cl2O PeCDF

354.9792 Lock C9F13 PFK

355.8546 M+2 C12H3

35Cl437ClO2

PeCDD

357.8516 M+4 C12 H3

35Cl337Cl2O2

PECDD

367.8949 M+2 13C12H3

35Cl437ClO2

PeCDD

369.8919 M+4 13C12H3

35Cl337Cl2O2

PeCDD

409.7974 M+2 C12H3

35Cl637ClO HpCDPE

3 373.8208 M+2 C12H2

35Cl537ClO HxCDF

375.8178 M+4 C12H2

35Cl437Cl2O HxCDF

383.8639 M 13C12H2

35Cl6O HxDDF

385.8610 M+2 13C12H2

35Cl537ClO HxCDF

389.8157 M+2 C12H2

35Cl537ClO2 HxCDD

391.8127 M+4 C12H2

35Cl437Cl2O2 HxCDD

392.9760 Lock C9F15 PFK

401.8559 M+2 13C12H2

35Cl537ClO2 HxCDD

403.8529 M+4 13C12H2

35Cl437Cl2O2 HxCDD

430.9729 QC C9F17 PFK

445.7555 M+4 C12H2

35Cl637Cl2O OCDPE

145
Table 4. Descriptors, exact m/z's, m/z types, and elemental compositions of the CDDs and

CDFs (cont.).

Descriptor Exact m/z* m/z type Elemental composition Substance

4 407.7818 M+2 C12H35Cl6

37ClO HpCDF

409.7789 M+4 C12H35Cl5

37Cl2O HpCDF

417.8253 M 13C12H35Cl7O HpCDF

419.8220 M+2 13C12H35Cl6

37ClO HpCDF

423.7766 M+2 C12H35Cl6

37ClO2 HpCDD

425.7737 M+4 C12H35Cl5

37Cl2O2 HpCDD

430.9729 Lock C9F17 PFK

435.8169 M+2 13C12H35Cl6

37ClO2 HpCDD

437.8140 M+4 13C12H35Cl5

37Cl2O2 HpCDD

479.7165 M+4 C12H35Cl7

37Cl2O NCDPE

5 441.7428 M+2 C12

35Cl737ClO OCDF

442.9728 Lock C10 F17 PFK

443.7399 M+4 C12

35Cl637Cl2O OCDF

457.7377 M+2 C12

35Cl737ClO2 OCDD

459.7348 M+4 C12

35Cl637Cl2O2 OCDD

469.7779 M+2 13C12

35Cl737ClO2 OCDD

471.7750 M+4 13C12

35Cl637Cl2O2 OCDD

513.6775 M+4 C12

35Cl837Cl2O DCDPE

* Nuclidic masses used:

H = 1.007825 O = 15.994915

C = 12.0000 35Cl = 34.968853

13C = 13.003355 37Cl = 36.965903

F = 18.9984

DCDPE = Decachlorodiphenyl ether OCDD = Octachlorodibenzo-p-dioxin

HpCDD = Heptachlorodibenzo-p-dioxin OCDF = Octachlorodibenzofuran

HpCDF = Heptachlorodibenzofuran OCDPE = Octachlorodiphenyl ether

HpCDPE = Heptachlorodibenzofuran PeCDD = Pentachlorodibenzo-p-dioxin

HxCDD = Hexachlorodibenzo-p-dioxin PeCDF = Pentachlorodibenzofuran

HxCDF = Hexachlorodibenzofuran PFK = Perfluorokerosene

HxCDPE = Hexachlorodiphenyl ether TCDD = Tetrachlorodibenzo-p-dioxin

NCDPE = Nonachlorodiphenyl ether TCDF = Tetrachlorodibenzofuran

Labeled compound.

There is only one m/z for 37Cl4-2,3,7,8-TCDD (cleanup standard)

146
Table 5. GC retention time window defining solution and isomer specificity test standard

(Section 3.1.2).

DB-5 column GC retention-time window defining solution

CDD/CDF First eluted Last eluted

TCDF 1,3,6,8- 1,2,8,9-

TCDD 1,3,6,8- 1,2,8,9-

PeCDF 1,3,4,6,8- 1,2,3,8,9-

PeCDD 1,2,4,7,9- 1,2,3,8,9-

HxCDF 1,2,3,4,6,8- 1,2,3,4,8,9

HxCDD 1,2,4,6,7,9- 1,2,3,4,6,7

HpCDF 1,2,3,4,6,7,8- 1,2,3,4,7,8,9

HpCDD 1,2,3,4,6,7,9- 1,2,3,4,6,7,8-

DB-5 column TCDD specificity test standard

1,2,3,7- and 1,2,3,8-TCDD

2,3,7,8-TCDD

1,2,3,9-TCDD

DB-225 column TCDF isomer specificity test standard

2,3,4,7-TCDF

2,3,7,8-TCDF

1,2,3,9-TCDF

The GC column performance check solution (CPSM/WDM, see Table 5) also contained the known

first and last PCDD/PCDF eluters under the conditions specified in this method. Their retention

times were used to determine the eight homologue retention time windows that were used for

qualitative and quantitative purposes. The retention times for the switching of ions

characteristic of one homologous series to the next higher homologous series was indicated in

the selected ion current profile (SICP). Accurate switching at the appropriate times was

absolutely necessary for accurate monitoring of these compounds.

2.2. Analyte identification criteria

2.2.1. Retention times

For 2,3,7,8-substituted congeners with an isotopically labeled quantitation or internal standard

present in the sample extract the retention time at maximum peak height of the sample

components (i.e., the two ions used for quantitation purposes listed in Table 4) were within -1

to +3 sec of the isotopically labeled standard.

For 2,3,7,8-substituted compounds that did not have an isotopically labeled quantitation or

internal standard present in the sample extract, the retention time fell within 0.005 retention

time units of the relative retention times measured in the routine calibration. Identification of

147
OCDF was based on its retention time relative to 13C12-OCDD as determined from the daily

routine calibration results.

The ion current responses for both ions used for quantitative purposes reach their maxima

within ±2 sec of each other.

The ion current responses for both ions used for the labeled standards reach their maxima

within ±2 sec of each other.

2.2.2. Ion abundance ratios

The integrated ion current profiles for the two ions used for quantitation purposes had a ratio

between the lower and upper limits established for the homologous series to which the peak was

assigned. See Table 6 for the required theoretical ion ratios and their QC acceptance limits.

2.2.3. Signal-to-noise ratio

All ion current intensities were greater than or equal to 3 times the noise level to be

considered positive identification of a PCDD or PCDF compound or a group of coeluting isomers.

The peaks representing the PCDDs and PCDFs and labeled compounds in the CS1 calibration

solution had signal-to-noise (S/N) ratios greater than or equal to 10.

2.2.4. Polychlorinated diphenylether interferences

In addition to the above criteria, the identification of a GC peak as a PCDF was only made if no

signal having a S/N greater than 3 was detected at the same retention time (±2 sec) in the

corresponding polychlorinated diphenylether (PCDPE, see Table 4) channel.

2.3. Calibration criteria

2.3.1. Initial calibration

All five calibration solutions listed in Table 3 (CS1-CS5) were used for the initial calibration.

The theoretical ion abundance ratios for all 17 native analytes and all 16 labeled compounds

were within the QC control limits specified in Table 6. The signal-to-noise ratio for the GC

signals present in each SICP (including the ones for the labeled standards) were greater than or

equal to 10. The percent relative standard deviations for the mean relative response factors

from the 17 unlabeled standards did not exceed ±20%, and those for the fifteen labeled

reference compounds did not exceed ±30%.

2.3.2. Continuing calibration (VER for EPA Method 1613)

Continuing calibration was performed at the beginning of each 12 hr period after successful

mass resolution and GC resolution performance checks were completed. A routine calibration

was also required at the end of a 12 hr run.

148
Table 6. Theoretical ion abundance ratios and QC limits*.

Number of m/z's forming ratio Theoretical ratio Lower Upper

chlorine atoms

4 M/(M+2) 0.77 0.65 0.89

5 (M+2)/(M+4) 1.55 1.32 1.78

6 (M+2)/(M+4) 1.24 1.05 1.43

6 M/(M+2) 0.51 0.43 0.59

7 (M+2)/(M+4) 1.05 0.88 1.20

7 • M/(M+2) 0.44 0.37 0.51

8 (M+2)/(M+4) 0.89 0.76 1.02

* QC limits represent ±15% windows around the theoretical ion abundance ratios.

Does not apply to 37Cl4-2,3,7,8-TCDD (cleanup standard).

Used for 13C12-HxCDF only.

• Used for 13C12-HpCDF only.

The isotopic ratios for all 17 native analytes and all 16 labeled compounds were within the

control limits for the theoretical ion abundance ratios specified in Table 6.

The signal-to-noise ratio for the GC signals present in each SICP (including the ones for the

labeled standards) were greater than 10.

The QC acceptance criteria for the calculated relative response factors for the unlabeled

standards were within ±20% of the mean values established during the initial calibration. QC

acceptance criteria for the calculated relative response factors for the labeled standards were

within ±30% of the mean values established during the initial calibration.

2.4. Criteria for QC samples in an analytical batch

2.4.1. Method blank

A Method Blank was used to demonstrate freedom from contamination in the analytical

procedure, and was required for each set of 20 or fewer samples.

If any of the 2,3,7,8-substituted PCDDs or PCDFs were found in the blank at greater than the

minimum level (Table 2), or if any potentially interfering compound is found in the blank at the

minimum level for each level of chlorination given in Table 2, re-extraction of the analytical

batch was indicated.

Analyst discretion was used when contamination was present which did not adversely effect

the analytical results.

If any of the 2,3,7,8-substituted PCDDs or PCDFs were found in the blank at greater than the

minimum level, but were not detected in the analytical samples above this level, no action was

required.

149
When target analytes were present in the method blank and in the analytical samples at

concentrations above the Minimum Level, analytical data for those analytes in the samples

were flagged.

2.4.2. Laboratory blank spike

A Laboratory Blank Spike (LBS) was used to demonstrate analytical precision of the method,

and was required with each set of 20 or fewer samples.

QC acceptance criteria for analyte recoveries was 70 to 130% of the spiked amount.

If six or more of the seventeen target analytes were outside the acceptance criteria corrective

action was taken. Corrective action may have included recalculation, reanalysis, instrument

maintenance, recalibration, or re-extraction of the analytical batch.

2.4.3. Matrix spike and matrix spike duplicate

A matrix spike (MS) and a matrix spike duplicate (MSD) sample were used to demonstrate

analytical accuracy and precision in the presence of a representative matrix and was required

for each set of 20 or fewer samples.

QC acceptance criteria for analyte recoveries in the MS and the MSD was 70 to 130% of the

spiked amount.

The results obtained from the MS and MSD samples should agree within a relative standard

deviation (RSD) of 20%.

The MS and MSD acceptance criteria were advisory. However, if six or more of the seventeen

target analytes were outside the QC acceptance criteria corrective action was taken.

Corrective action may have included recalculation, reanalysis of the MS and MSD, instrument

maintenance, or recalibration.

2.4.4. Duplicate

A sample duplicate (DUP) was used to demonstrate matrix homogeneity and analytical precision

in the presence of a representative matrix, and was required with each set of 20 or fewer

samples.

QC acceptance criteria for analyte concentrations greater than ten times the minimum level

was a RSD of 20% or less.

The DUP acceptance criteria were advisory. However, if six or more of the seventeen target

analytes were outside the QC acceptance criteria corrective action was taken. Corrective

action may have included recalculation, reanalysis, instrument maintenance, or recalibration.

2.4.5. Reference material

A Reference Material (RM) was used to demonstrate analytical accuracy on a material from an

independent source and was required with each set of 20 or fewer samples.

QC acceptance criteria for analyte concentrations less than ten times the ML were ±35% of the

true value.

150
The QC acceptance criteria was invalid when the analyte concentration falls below the minimum

level listed in Table 2.

The SRM acceptance criteria were advisory. However, if more than 35% of the analytes fall

outside the QC acceptance criteria, corrective action was taken. Corrective action included

recalculation and/or reanalysis of the SRM, instrument maintenance and/or recalibration.

2.4.6. Labeled compound recovery

All samples were spiked with the Labeled Compound Spiking Solution (LCSS) to monitor method

performance on the sample matrix. QC acceptance criteria for labeled compound was 40% to

135% recovery. If a labeled compound recovery fell below the acceptance criteria, but the sum

of the areas of the affected peaks was 10% or greater than the area of the corresponding peaks

in the continuing calibration standard, the analytical data were accepted. If six or more of the

fifteen labeled compounds were outside the acceptance criteria corrective action was taken.

Corrective action may have included recalculation, reanalysis, instrument maintenance,

recalibration, or re-extraction of the sample aliquot.

3. CHROMATOGRAPHIC CONDITIONS

3.1. Gas chromatograph

The gas chromatograph (GC) used for this analytical method was an HP5890 Series II. The GC

had an injection port designed for capillary columns and splitless injections, was capable of

temperature ramp programming with an isothermal hold, and of meeting all of the performance

specifications outlined in Section 2.1.2. A 2 μL splitless injection was used for all extracts,

blanks, calibration solutions and the performance check samples.

All injections were made with a CTC-2000S programmable autosampler.

The GC/MS interface components withstand 350 °C, and were designed so that the separation

of 2,3,7,8-TCDD from the other TCDD isomers achieved in the gas chromatographic column

was not appreciably degraded. The GC column was fitted directly into the mass spectrometer

ion source without being exposed to the ionizing electron beam.

3.2. GC columns

The GC column used for analysis of PCDDs and PCDFs and for isomer specificity of 2,3,7,8-

TCDD was a 60 meter J&W DB5 or DB5MS column with a 0.25 mm i.d. and a 0.25 μm film

thickness.

The GC column used for isomer specificity and confirmation of 2,3,7,8-TCDF was a J&W

DB225 column with 0.25 mm i.d. and 0.25 μm film thickness.

3.3. Operating conditions

Operating conditions known to produce acceptable results with the recommended analytical

column (DB5MS) are shown below:

Inject at 190 °C, hold for 1 min

Ramp at 20 °C per min to 240 °C

Ramp at 1 °C per min to 295 °C, hold for 2 min

151
Operating conditions known to produce acceptable results with the recommended confirmation

column (DB225) are shown below:

Inject at 200 °C, hold for 1 min

Ramp at 12 °C per min to 300 °C, hold for 10 min

4. DETECTOR AND DATA SYSTEM CRITERIA

The detector used for these analyses was a VG AutoSpec Ultima utilizing 35 eV electron impact

ionization, capable of repetitively selectively monitoring a minimum of 12 exact m/z's at high

resolution (>10,000) during a period of approximately 1 sec or less, and meeting all of the

performance specifications outlined in Section 3.2.1.

A dedicated data system was employed to control the rapid multiple-ion monitoring process and

to acquire the data. Quantitation data (peak areas and/or peak heights) and Selected Ion

Monitoring (SIM) traces are acquired during the analyses and stored. The data system was

capable of acquiring data at a minimum of 12 ions in a single scan. It was capable of switching

to different sets of ions (descriptors) at specified times during an acquisition, and of providing

hard copies of individual ion chromatograms for selected gas chromatographic time intervals. It

was capable of acquiring mass spectral peak profiles and providing hard copies of peak profiles

to demonstrate the required resolving power. The data system permitted the measurement of

noise on the base line.

5. INSTRUMENT CALIBRATION PROCEDURE

5.1. Initial calibration

Initial calibration was required before any samples were analyzed for PCDDs and PCDFs. Initial

calibration was also required if any continuing calibration verification did not meet the required

criteria listed in Section 3.3.2. All five high resolution concentration calibration solutions listed

in Table 3 were used for the initial calibration.

The instrument was tuned with PFK as described in Section 2.1.1.

The CPSM/WDM (Table 5) was injected and the SIM mass spectral data were acquired as

described in Section 2.1.1. The criteria listed in Section 2.2.2 were met.

Using the same GC and MS conditions that produced acceptable results with the CPSM/WDM, a 2

μL portion of each of the five calibration solutions (CS1 - CS5) were analyzed and the following

conditions were met.

The ratio of integrated ion currents for the homologous series quantitation ions appearing in

Table 6 were within the indicated control limits set for each homologous series.

The ratio of integrated ion currents for the ions belonging to the 13C12-labeled quantitation and

internal standards were within the control limits stipulated in Table 6.

All ratios were within the specified control limits simultaneously in one run. Otherwise,

corrective action was necessary.

152
For each SICP and for each GC signal corresponding to the elution of a target analyte and of its

13C12-labeled standard(s), the signal-to-noise (S/N) ratio was greater than or equal to 10.

Referring to Table 2, the 17 relative response factors (RRF) for unlabeled target analytes

[RRF(n); n = 1 to 17] relative to their appropriate quantitation standards, and the RRFs for the

13C12-labeled quantitation standards [RRF(m); m = 1 to 15] relative to the two internal

standards were calculated according to the following equations.

Eq. 2 RRF(n) = 

Ax C qs

Aqs C x

Eq. 3 RRF(m) = 

Aqs C is

Ais C qs

where Ax was the sum of the integrated ion abundances of the quantitation ions for unlabeled

PCDDs and PCDFs, Aqs was the sum of the integrated ion abundances of the quantitation ions for

the labeled quantitation standards, Ais was the sum of the integrated ion abundances of the

quantitation ions for the labeled internal standards, Cx was the concentration of the unlabeled

PCDD and/or PCDF analyte in the calibration solution (pg/μL), Cqs was the concentration of the

13C12-labeled quantitation standard in the calibration solution (100 pg/μL), and Cis was the

concentration of the 13C12-labeled internal standard in calibration solution (100 pg/μL). The

RRF(n) and RRF(m) are dimensionless quantities. The units used to express Cx, Cqs and Cis were

the same.

The native OCDF was quantitated against labeled OCDD. The concentration of OCDF was

corrected for the recovery of the 13C12-OCDD.

The 1,2,3,7,8,9-HxCDD isomer was quantitated using the averaged response of the labeled

analogs of the other two 2,3,7,8-substituted HxCDDs. As a result, the concentration of

1,2,3,7,8,9-HxCDD was corrected for the average recovery of the other two 13C12-HxCDDs.

The mean RRFs, RRF(n) , for the five calibrations solutions were calculated using the following

equations.

Eq. 4 RRF(n) = 

1

5

j=1

5

 RRFj(n)

Eq. 5 RRF(m) = 

1

5

j=1

5

 RRFj(m)

where n was a particular 2,3,7,8-substituted PCDD/PCDF unlabeled target analyte (n = 1 to

17), m was a particular 13C12-labeled quantitation standard (m = 1 to 15), and j was the

injection number or calibration solution number (j = 1 to 5).

The percent relative standard deviations (%RSD) for each calibration mixture was determined.

It was verified that the initial calibration met all of the acceptance criteria outlined in Section

2.3.1.

153
5.2. Continuing calibration verification

Routine calibration was performed at the beginning of a 12 hr period after successful MS

resolution and GC resolution performance checks. A routine calibration was also required at the

end of a 12 hr shift.

Using the same GC and MS conditions that were used for the CPSM/WDM and the initial

calibration, a 2 μL portion of the midpoint calibration solution (CS3) was analyzed with the

following mass spectrometer operating parameters.

The ratio of integrated ion currents for the homologous series quantitation ions appearing in

Table 6 were within the indicated control limits set for each homologous series.

The ratio of integrated ion currents for the ions belonging to the 13C12-labeled quantitation and

internal standards were within the control limits stipulated in Table 6.

All ratios were within the specified control limits simultaneously in one run. Otherwise,

corrective action was necessary.

For each SICP and for each GC signal corresponding to the elution of a target analyte and of its

13C12-labeled standard(s), the signal-to-noise (S/N) ratio was greater than or equal to 10.

Referring to Table 2, the concentrations of the 17 unlabeled target analytes, and the

concentration and percent recovery of the fifteen 13C12-labeled quantitation standards in the

CS3 standard were calculated using the following equations.

Eq. 6 Cx = 

Ax C qs

Aqs RRF(n)

Eq. 7 Percent recovery = 

Ax C is

Ais C qs RRF(m)

 100

where Ax was the sum of the integrated ion abundances of the quantitation ions for unlabeled

PCDDs and PCDFs, Aqs was the sum of the integrated ion abundances of the quantitation ions for

the 13C12-labeled quantitation standards, Ais was the sum of the integrated ion abundances of

the quantitation ions for the 13C12-labeled internal standards, Cx was the concentration of the

unlabeled PCDDs and PCDFs isomers in pg/μL, Cqs was the concentration of the 13C12-labeled

quantitation standard in the calibration solution (100 pg/μL), Cis = concentration of the 13C12-

labeled internal standard in the calibration solution (100 pg/μL), RRF(n) was the mean

relative response factor for the unlabeled target analyte relative to its 13C12-labeled

quantitation standard [RRF(n), with n = 1 to 17], and RRF(m) was the mean relative response

factor for 13C12-labeled quantitation standard relative to its 13C12-labeled internal standard

[ RRF(m) , with m = 1 to 15].

Referring to Table 2, the 17 relative response factors (RRF) for unlabeled target analytes

(RRF(n)); n = 1 to 17] relative to their appropriate quantitation standards (Eq. 2, Section

6.1.4), and the RRFs for the 13C12-labeled quantitation standards [(RRF(m)); m = 1 to 15]

relative to the two internal standards (Eq. 3, Section 5.1.4) were calculated.

154
6. ANALYTICAL STANDARDS

Analytical standards were purchased as solutions with certified purity, concentration and

authenticity. The labeled compound spiking solution (LCSS), the cleanup recovery standard

(CRS), the precision and recovery standard (PAR), the internal standard (IS) and the

instrument calibration solutions (CS1 - CS5) were used as received from the manufacturer

without further treatment. When not being used, standards were stored in the dark at 40 °C in

screw-capped vials with PTFE-lined caps.

6.1. Labeled compound spiking solution

The labeled compound spiking solution (LCSS) solution contained the fifteen 13C12-labeled PCDD

and PCDF quantitation standards in nonane at the nominal concentrations listed in Table 7. The

LCSS (20 μL) was diluted in an appropriate solvent and spiked into each sample prior to

extraction.

6.2. Precision and recovery standard

The precision and recovery standard (PAR) solution contained the seventeen 2,3,7,8-

substituted native PCDD and PCDF isomers at the nominal concentrations listed in Table 7. This

solution (5 μL) was diluted in an appropriate solvent and spiked into the selected matrix spike

(MS), matrix spike duplicate (MSD), and/or laboratory blank spike (LBS) samples prior to

extraction.

6.3. Internal standard

This solution contained two 13C12-labeled PCDD isomers. The internal standard (IS) (10 μL)

was added to the final sample extract before HRGC/HRMS analysis to determine the percent

recoveries for the LCSS and CRS compounds.

6.4. Instrument calibration standards

These solutions contained the seventeen 2,3,7,8-substituted native PCDDs and PCDFs and the

sixteen 13C12-labeled quantitation and internal standards at the nominal concentrations listed in

Table 3. The analyte concentrations were homologue dependent, with the lowest concentrations

for the tetrachlorinated dioxin and furan and the highest concentrations for the octachlorinated

congeners. These solutions permitted the relative response factors to be measured as a

function of concentration. The CS3 standard was also used as the continuing calibration

verification (VER).

6.5. Column performance and window defining mix

This solution contained the first and last eluting isomers for each homologous series from

tetra- through heptachlorinated congeners. The solution also contained a series of other TCDD

isomers for the purpose of documenting the chromatographic resolution of 2,3,7,8-TCDD. The

13C12-2378-TCDD was present for positive identification of the 2,3,7,8-TCDD isomer (see

Table 5).

155
6.6. 2,3,7,8-TCDF CPSM

This solution contained a series of three TCDF isomers for the purpose of documenting the

chromatographic resolution of 2,3,7,8-TCDF on a DB225 analytical column (see Table 5).

7. SAMPLE ANALYSIS

The instrument was tuned with PFK as described in Section 2.1.1.

The CPSM/WDM (Table 5) was injected and the SIM mass spectral data was acquired as

described in Section 2.1.1.

The CS3 Calibration Verification solution (Table 3) was injected and the SIM mass spectral data

were acquired as described in Section 2.1.1. The acceptance criteria listed in Section 2.3.2

were met.

Toluene (2 μL) was injected and the SIM mass spectral data was acquired to demonstrate that

the. analytical system was free of interfering contamination.

The sample extract (2 μL) was injected and the SIM mass spectral data acquired under these

same conditions.

7.1. Qualitative identification

For a gas chromatographic peak to be identified as a PCDD or PCDF isomer, it met all of the

criteria specified in Section 3.2.

7.2. Quantitative determination

For gas chromatographic peaks that met all the qualitative identification criteria, the

concentration of the PCDD or PCDF isomers were calculated using the following equation:

Eq. 8 Cx = 

Ax C qs

Aqs W RRF(n)

where Cx was the concentration of the unlabeled PCDD and PCDF isomer in pg/g (ppt), Ax was

the sum of the integrated ion abundances of the quantitation ions for unlabeled PCDDs and

PCDFs, Aqs was the sum of the integrated ion abundances of the quantitation ions for the 13C12-

labeled quantitation standards, Qqs was the amount in pg of the 13C12-labeled quantitation

standard added to the sample immediately prior to extraction, RRF(n) was the mean relative

response factor for the unlabeled target analyte relative to its 13C12-labeled quantitation

standard [ RRF(n) , with n = 1 to 17], and W was the weight, in g, of the sample.

The percent recovery of the fifteen quantitation standards in the sample extract was calculated

using the following equation:

Eq. 9 Percent recovery = 

Aqs Qis

Ais Qqs RRF(m)

 100

156
where Aqs was the sum of the integrated ion abundances of the quantitation ions for the 13C12-

labeled quantitation standards, Ais was the sum of the integrated ion abundances of the

quantitation ions for the 13C12-labeled internal standards, Qqs was the amount in pg of the

13C12-labeled quantitation standard added to the sample immediately prior to extraction, Qis

was the amount in pg of the 13C12-labeled internal standard added to the sample extract in the

last step of preparation, RRF(m) was the mean relative response factor for 13C12-labeled

quantitation standard relative to its 13C12-labeled internal standard [ RRF(m) , with m = 1 to

15].

A sample specific estimated detection limit (EDL) was calculated for each 2,3,7,8-substituted

congener that was not identified, regardless of whether or not other non-2,3,7,8-substituted

isomers were present. The EDL was the concentration of a given analyte required to produce a

signal with a peak height of at least 3 times the background signal level. The quantitation

software (OPUSQuan) supplied by the instrument manufacturer calculated a sample specific-EDL

using an algorithm based on the criteria outlined in EPA Method 8290, Section 7.9.5.1.1.

The relative percent difference (RPD) between duplicate sample results was calculated.

The percent recovery of analytes in the MS and MSD samples were reported.

7.3. Confirmation analysis

If the 2,3,7,8-TCDF isomer was identified at a concentration which was above the minimum

level, the 2,3,7,8-TCDF concentration was confirmed on a second analytical column (DB225).

Following the steps outlined in 7.1 through 7.6, the sample extract was reanalyzed on a DB225

analytical column. Use the GC conditions described in Section 3.3.2, and the mass spectral

conditions described in section 2.1.1, only the ions listed in the first descriptor (Table 4) were

monitored.

The concentration of 2,3,7,8-TCDF were calculated using Equation 8.

The lower of the two calculated concentrations was reported for 2,3,7,8-TCDF (DB5 and

DB225).

8. INSTRUMENT MAINTENANCE

8.1. Gas chromatograph maintenance

The syringe was cleaned by rinsing with appropriate solvent after each injection. A new

injection port liner and septum were installed at the beginning of each new run sequence. A new

injection port base plate was installed as needed when chromatographic resolution fell below

the QC acceptance criteria. One to two feet of the analytical column were removed as needed

when chromatographic resolution fell below the QC acceptance criteria. The tank of carrier gas

(He) was replaced when the pressure falls below 500 psi. All maintenance was recorded.

8.2. Mass spectrometer maintenance

The emission filament was replaced as necessary. The inner ion source assembly was cleaned

and replaced as necessary. The outer ion source assembly was cleaned and replaced as

necessary. The septum on the PFK reservoir was replaced as necessary. The transfer line/re157
entrant assembly was disassembled, cleaned, repaired and reassembled as necessary. The

rotary pump oil was changed yearly, or more frequently if indicated. The diffusion pump oil

was changed as necessary. All maintenance was recorded.

9. CONCLUSIONS

This method provides procedures for the detection and quantitative measurement of

polychlorinated dibenzo-p-dioxins (tetra- through octa-chlorinated homologues; PCDDs) and

polychlorinated dibenzofurans (tetra- through octa-chlorinated homologues; PCDFs) in

sediments and bivalves at pg/g concentrations. The sensitivity of this method is dependent upon

the level of interferences within a given matrix. The calibration range of the method for a 10 g

sediment or tissue sample is 1.0 to 400 ppt for TCDD/TCDF, 5.0 to 2,000 ppt for

PeCDD/PeCDF, HxCDD/HxCDF and HpCDD/HpCDF, and 10 to 4,000 ppt for OCDD/OCDF. The

actual limits of detection will differ from the lower method calibration limit, depending on the

complexity of the matrix.

10. REFERENCES

EPA (1992) EPA Method 8290: Polychlorinated Dibenzodioxins (PCDDs) and Polychlorinated

Dibenzofurans (PCDFs) by High-Resolution Gas Chromatography/High Resolution Mass

Spectrometry (HRGC/HRMS), Revision 0, SW-846, U.S. Environmental Protection Agency,

Office of Solid Waste, November 1992.

EPA (1994) EPA Method 1613: Tetra- Through Octa-Chlorinated Dioxins and Furans by Isotope

Dilution HRCG/HRMS, Revision B, EPA 821-B-94-005, U.S. Environmental Protection Agency,

Office of Water, October 1994.

158
Table 7. Concentration of stock and spiking solutions containing PCDDs and PCDFs labeled

compounds.

CDD/CDF Labeled Compound spiking

solution (LCSS) PAR spiking solution

(pg/μL) (pg/μL)

2,3,7,8-TCDD ---- 40

2,3,7,8-TCDF ---- 40

1,2,3,7,8-PeCDD ---- 200

1,2,3,7,8-PeCDF ---- 200

2,3,4,7,8-PeCDF ---- 200

1,2,3,4,7,8-HxCDD ---- 200

1,2,3,6,7,8-HxCDD ---- 200

1,2,3,7,8,9-HxCDD ---- 200

1,2,3,4,7,8-HxCDF ---- 200

1,2,3,6,7,8-HxCDF ---- 200

1,2,3,7,8,9-HxCDF ---- 200

2,3,4,6,7,8-HxCDF ---- 200

1,2,3,4,6,7,8-HpCDD ---- 200

1,2,3,4,6,7,8-HpCDF ---- 200

1,2,3,4,7,8,9-HpCDF ---- 200

OCDD ---- 400

OCDF ---- 400

13C12-2,3,7,8-TCDD 100 ----

13C12-2,3,7,8-TCDF 100 ----

13C12-1,2,3,7,8-PeCDD 100 ----

13C12-1,2,3,7,8-PeCDF 100 ----

13C12-2,3,4,7,8-PeCDF 100 ----

13C12-1,2,3,4,7,8-HxCDD 100 ----

13C12-1,2,3,6,7,8-HxCDD 100 ----

13C12-1,2,3,4,7,8-HxCDF 100 ----

13C12-1,2,3,6,7,8-HxCDF 100 ----

13C12-1,2,3,7,8,9-HxCDF 100 ----

13C12-2,3,4,6,7,8-HxCDF 100 ----

13C12-1,2,3,4,6,7,8-HpCDD 100 ----

13C12-1,2,3,4,6,7,8-HpCDF 100 ----

13C12-1,2,3,4,7,8,9-HpCDF 100 ----

13C12-OCDD 200 ----

Concentration (pg/μL)

Cleanup standard 37Cl4-2,3,7,8-TCDD 0.8

Internal standards 13C12-1,2,3,4,-TCDD 200

13C12-1,2,3,7,8,9-HxCDD 200

159
Quantitative Determination of Chlorinated Hydrocarbons

J. L. Sericano, P. Gardinali, and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

Chlorinated hydrocarbons are quantitatively determined by gas chromatography with an

electron capture detector (ECD). The ECD is very sensitive and allows for detection of

the chlorinated hydrocarbons at trace concentrations (ppb).

1. INTRODUCTION

The quantitative method described in this document determines chlorinated hydrocarbons (e.g.

chlorinated pesticides and PCBs) in sample extracts. The method is based on high resolution,

capillary gas chromatography using electron capture detection (GC/ECD).

Sample collection, preservation, storage and holding times are discussed under the analytical

procedures for sample extraction and purification.

2. APPARATUS AND MATERIALS

A gas chromatograph with a split/splitless injection system, capillary column capability and a

electron capture detector (ECD) was utilized.

2.1. GC Column

A 30-m long x 0.25-mm i.d. fused silica capillary column with DB-5 and DB-17 HT

(confirmation column) bonded phase (J&W Scientific or equivalent) were used. The column

provided good resolution of chlorinated hydrocarbons, surrogates and internal standards.

2.2. Autosampler

The autosampler was capable of making 1 - 4 μL injections.

3. REAGENTS

3.1. Calibration Solution

The calibration solution was comprised of the chlorinated hydrocarbons indicated in Table 1.

160
Table 1. Chlorinated hydrocarbons of interest.

Chlorinated pesticides Polychlorinated biphenyls

1,2,4,5-Tetrachlorobenzene * Dichlorobiphenyls

1,2,3,4-Tetrachlorobenzene * PCB 8*

Pentachlorobenzene*

Pentachloroanisole* Trichlorobiphenyls

Endosulfan II PCB 18*, PCB 31, PCB 28*

alpha-HCH*

Hexachlorobenzene* Tetrachlorobiphenyls

beta-HCH* PCB 52*, PCB 49, PCB 44*, PCB 74, PCB

gamma-HCH* 70, PCB 66*, PCB 60

delta-HCH*

Heptachlor* Pentachlorobiphenyls

Heptachlor Epoxide* PCB 84, PCB 85, PCB 101*, PCB 87*, PCB

99, PCB 97, PCB 110, PCB 123, PCB 118*,

PCB 114, PCB 105*

Oxychlordane*

gamma-Chlordane*

alpha-Chlordane*

trans-Nonachlor* Hexachlorobiphenyls

cis-Nonachlor* PCB 153*, PCB 138*, PCB 158, PCB 128*,

Aldrin* PCB 167, PCB 156, PCB 157

Dieldrin*

Endrin* Heptachlorobiphenyls

Mirex* PCB 187*, PCB 183, PCb 174, PCB 177, PCB

2,4'-DDE* 180*, PCB 170*, PCB 189

4,4'-DDE*

2,4'-DDE* Octachorobiphenyls

4,4'-DDE*

2,4'-DDT* PCB 201, PCB 196, PCB 195*, PCB 194,

4,4'-DDT* PCB 203

Nonachlorobiphenyl

PCB 206*

Decachlorobiphenyl

PCB 209*

*Analytes in matrix spiking solution.

PCB number from Ballschmiter and Zell (1980).

Calibration standards were prepared in the concentration range of 5 to 200 ng/mL at four

concentrations. Internal standard and surrogate compounds were added at 100 ng/mL to all

calibration standards.

3.2. Surrogate spiking solution

The surrogate compounds for all sample types were 4,4'-dibromooctafluorobiphenyl (DBOFB),

PCB 103, and PCB 198. A surrogate solution was made by weighing an appropriate aliquot of

161
pure material into a volumetric flask and diluting to volume with hexane. Surrogate standards

were added to each sample at a concentration greater than ten times the MDL.

3.3. Internal standard solution

The internal standard for this analysis was tetrachloro-m-xylene (TCMX). An internal standard

solution was made by weighing an appropriate aliquot of pure material into a volumetric flask

and diluting to volume with hexane. Internal standard were added to each sample extract to

obtain a final concentration of approximately 100 ng/mL.

3.4. Matrix recovery spiking solution

The matrix spike was added to samples at a concentration of 40 ng/mL.

3.5. Retention index solution

The calibration mixtures were used to determine retention windows. Retention windows were

±0.07 min for all analytes in Table 1, with the exception of PCB 209, which had a retention

window of ±0.10 min.

4. PROCEDURE

4.1. Sample extraction and purification

Samples were extracted and purified following methods described in other sections of this

document.

4.2. High resolution GC-ECD analysis

4.2.1. GC conditions

For the analysis of chlorinated hydrocarbons, the analytical system, or its equivalent, included

at a minimum,

Instrument: Hewlett-Packard 5880A or 5890 or Varian 3500 series

Features: Split/splitless capillary inlet system, with data acquisition system

Inlet: Splitless

Detector: Electron Capture

Column: 0.25-mm i.d. x 30-m DB-5 fused

silica capillary column (J&W Scientific) or equivalent

Second Column: 0.25 mm i.d. x 30-m DB-17 HT fused silica capillary column (J&W

Scientific) or equivalent

Gases:

Carrier: Helium 1 mL/min

Make-Up: Argon/methane (95/5) or nitrogen, 40 mL/min.

162
Temperatures:

Injection port: 275 °C

Detector: 325 °C

Oven Program: 100 °C for 1 min., then 5 °C/min. to 140 °C, hold 1 min.; 1.5 °C/min

to 250 °C, hold 1 min.; 10 °C/min to 300 °C, hold 5 min.

The GC oven temperature program may be modified to improve

resolution.

Calibration: Four-point calibration (5, 20, 80, and 200 ng/mL)

Quantification: Surrogate standard/calibration

4.2.2. Calibration

Pesticide/PCB calibration was done as part of the analytical run. The four calibration mixtures

were interspersed with actual samples during the GC/ECD analyses. The calibration curve was

then based on four standards. If the calibration curve had an r of 0.9950 or higher for all

analytes present in the samples it was accepted, if not the calibration standards as well as all

the samples were reanalyzed by GC/ECD. Calibrations with internal standards was superior to

the procedure where the instrument was initially calibrated at four points and then mid-level

standards were analyzed during the analytical run. This latter calibration only insures that

mid-level samples remain in calibration. Since the ECD detector was nonlinear, a single point

check on its calibration was not as rigorous as calibration during the GC/ECD run.

4.2.3. Sample Analysis

As discussed in Section 4.2.2 calibration mixture, actual samples, and QA samples (e.g.,

blanks, matrix spikes, SRM) were analyzed as one analytical sequence.

Sample injections of 1 to 4 μL were made with an autosampling device.

If the response for any peak exceeded the highest calibration solution, the extract was diluted,

a known amount of surrogate and TCMX solution added, and the sample reanalyzed for those

analytes that exceeded the calibration range. If dilutions were necessary, the final

concentration reported was adjusted to account for the surrogate recoveries determined by the

initial injections.

4.2.4. Calculations

Concentrations in the samples were based on surrogate standards added. Analyte

concentrations were calculated using the PCB 103 surrogate. The internal standard (TCMX) was

used to calculate surrogate recoveries. DBOFB or PCB 198 was used to calculate selected

analytes concentrations, if it was demonstrated that they produced more reliable data (i.e., if

matrix interference occurs with PCB 103) based on percent recoveries in spiked blanks,

matrix spikes, or reference materials.

163
5. QUALITY ASSURANCE/QUALITY CONTROL (QA/QC) REQUIREMENTS

5.1. Calibration checks

A four-point calibration curve was used to establish the response of the detector. The

calibration curve was prepared using a non-linear calibration equation of the form:

Y = b0 + b1x + b2x

2

where Y is the modified area ratio; the ratio of the area of the analyte to the area of the

internal standard times the amount of the internal standard in micrograms.

Y = 

A(a)

A(s) C(s)

and b0, b1, and b2 are coefficients of the quadratic equation.

A solution to the quadratic equation is:

X = 

b1 + b

2

1 - 4b 2

(b0 - Y)

2b2

which will give you the amount of analyte in μg/μL.

Alternatively, the following equation can be used:

Y = A xB

Y = 

Ca

Csu

 = A 

Aa

Asu

B

where, A is the slope of the line fit, B is the polynomial coefficient for the line fit, Ca is the

concentration of the analyte to be measured (ng/mL), Csu is the concentration of the surrogate

standard (ng/mL) (PCB 103), Aa is the area for the analyte to be measured and Asu is the area

for the surrogate standard (PCB 103).

The calibration solutions that were analyzed as part of the analytical GC/ECD run, were

preceded by no more than six samples and no more than six samples are run between

calibration mixtures.

5.2. Method blank analysis

An acceptable method blank contained no more than two target compounds at concentrations

three times greater than the MDL. This criteria did not need to be met if the analytes in the

blanks were not detected in the associated samples or the analyte concentration was greater

than ten times the blank value. If the method blank did not meet these criteria, the analytical

system was out of control, and the source of the contamination was investigated, corrective

measures taken, and samples reanalyzed.

164
5.3. Surrogate standard analysis

All samples and quality control samples were spiked with DBOFB, PCB 103 and PCB 198. The

surrogate standard solution was spiked into the sample prior to extraction in an attempt to

minimize individual sample matrix effects associated with sample preparation and analysis.

The laboratory took corrective action whenever the recovery of the surrogate used for

quantitation was outside of 50 to 125% range.

The following corrective action were taken when an out of control event occurs,

a. Calculations were checked to assure that no errors had been made.

b. The surrogate standard solutions were checked for degradation, contamination, or

concentration, and the instrument performance was checked.

c. If the surrogate could not be measured because the sample required dilution or only a

portion of the sample was analyzed, or matrix interference occurs with only one

surrogate, no corrective action was required. The surrogate recovery was properly

annotated.

d. If the steps above failed to reveal a problem, the sample or extract was reanalyzed. If

reanalysis of the extract yielded surrogate recoveries within the stated limits, then

the reanalyzed data were reported. If upon reinjection QA criteria were still violated,

the sample was submitted for re-extraction if sufficient sample was available. If the

sample was completely consumed, the data was reported but designated as outside the

QA criteria.

5.4. Matrix spike/duplicate analysis

A matrix spike and a duplicate were analyzed with each sample set or every 20 field samples,

whichever was more frequent. A sample was randomly chosen and split into subsamples. One

subsample was fortified with the matrix spike solution. The acceptable matrix spike recovery

criteria were 50 - 125% recovery for at least 80% of the analytes. Criterion for duplicates

was 30% RPD.

If the matrix spike criteria were not met, the matrix spike was reinjected on the GC. If the

reinjected matrix spike analysis met the criteria, then the reanalysis data was reported. If

none of the analytes that were in violation were present in the sample the violation was noted

but no action was required. If analytes that were present in the sample were in violation, the

entire batch of samples was submitted for re-extraction, if sufficient sample was available. If

the sample was completely consumed, the data was reported but designated as outside the QA

criteria.

5.5. Method detection limit

The method detection limit was determined following the procedures outlined in Federal

Register (1984), Vol. 49, No. 209, 198-199.

5.6. GC resolution

Most target compounds, surrogates and internal standard were resolved from one another and

from interfering compounds. When they were not, coelutions were documented.

165
5.7. Reference material analysis

Standard reference materials (SRM) were analyzed for chlorinated hydrocarbons. One SRM

sample was analyzed per batch of samples or every 20 samples whichever was more frequent.

Results were within ±30% of the certified or reference value on the average for all analytes,

and did not exceed ±35% of the certified or reference value for more than 30% of the

individual analytes. If a value was not certified the QC criteria (35% and 30%) apply to either

the upper or lower 95% confidence interval reference value.

6. CALCULATIONS

6.1. Chlorinated hydrocarbon calculations

All calculations were based on the surrogate PCB 103 added before extraction and purification.

The actual sample concentration (C) for each compound was calculated by the following

formula:

C = A 

Aa

Asu

B ISU

SDW

where A was the slope of the curve fit, B was the polynomial coefficient for the curve fit, Aa

was the area for the analyte to be measured, Asu is the area for the surrogate (PCB 103), Isu

was the amount of surrogate added to the sample, and SDW was the sample dry weight.

6.2. Calculation notes

To each sample, a specific amount of surrogate standard was added. The recovery of these

compounds was monitored in each sample using the response of TCMX the internal standard (Igc)

added to the final extract just prior to GC/ECD analyses.

Percent surrogate recovery = R1 R2 R3 R4 100

where R1 was the surrogate peak area/internal standard peak area in sample, R2 was the

surrogate concentration/internal standard concentration in one of the calibration mixtures, R3

was the internal standard peak area/surrogate peak area in one of the calibration mixtures, and

R4 was the a mount of internal standard (Igc) added to sample just prior to GC analysis/amount

of surrogate standard added to sample just prior to sample extraction.

7. REPORTING

Data were reported in ng/g dry weight. The minimum method performance standard for tissues

was 0.5 ng/g for individual compounds. Results were reported to three significant figures.

Surrogate recoveries were reported for each sample analyzed. Matrix spike recoveries and

duplicate relative percent difference were reported for each batch of samples analyzed. Results

of the analysis of reference materials were reported for each batch of samples analyzed.

166
8. CONCLUSIONS

The electron capture detector provides a means to detect and quantitate chlorine containing

organic contaminants at trace concentrations (ng/g). The detector, however, does not have a

linear response over a wide concentration range and must be used by sufficiently trained

personnel.

9. REFERENCE

Ballschmiter, K., and M. Zell (1980) Analysis of polychlorinated biphenyls (PCB) by glass

capillary gas chromatography. Frensenius Z. Anal. Chem., 302:20-31.

167
Quantitative Determination of Butyltins

J. L. Sericano and T. L. Wade

Geochemical and Environmental Research Group

Texas A&M University

College Station, TX

ABSTRACT

The quantitative determination of tetrabutyltin, tributyltin, dibutyltin, and

monobutyltin in sediment and tissue samples by high resolution, capillary gas

chromatography using flame photometric detection is described. Final concentrations

are reported as ng/Sn/g for sediment and tissue samples.

1. INTRODUCTION

The quantitative method described in this document determined butyltins in extracts of water,

sediments and tissues. The method was based on high resolution, capillary gas chromatography

using flame photometric detection (GC/FPD). This method quantitatively determined

tetrabutyltin (4BT), tributyltin (TBT), dibutyltin (DBT) and monobutyltin (MBT).

Sample collection, preservation and storage times are discussed under the analytical

procedures for sample extraction and purification.

2. APPARATUS AND MATERIALS

A gas chromatograph with a split/splitless injection system, capillary column capability and a

flame photometric detector (FPD) equipped with a tin selective 610 nm filter was used. The

output from the detector was collected and processed by an automated data acquisition

software package.

2.1. GC Column

A 30-m long x 0.32-mm i.d. fused silica capillary column with DB-5 bonded phase (J&W

Scientific or equivalent) was used.

2.2. Autosampler

The autosampler was capable of making 1 - 4 μL injections.

3. REAGENTS

3.1. Calibration solution

The calibration solution was comprised of 4BT, TBT, DBT and MBT.

The calibration standards were prepared in the concentration range of 1.25 to 50 ng Sn/mL (at

five concentrations) at a minimum. Internal standard and surrogate compounds were added at

20 ng Sn/mL to all calibration standards.

168
3.2. Surrogate spiking solution

The surrogate compounds for all sample types were tripropyltin chloride (TPT) and dimethyltin

dichloride (DMT). The surrogate solution was prepared by weighing an appropriate amount of

pure material into a volumetric flask and diluting to volume with dichloromethane. Surrogates

were added to each sample at a concentration of approximately ten times the MDL (i.e., 100 μL

of 20 ng Sn/mL to a final volume of 1 mL).

3.3. Internal standard solution

The internal standard for this analysis was tetrapropyltin (4PT). The internal standard solution

was prepared by weighing an appropriate amount of pure material into a volumetric flask and

diluted to volume with dichloromethane. The internal standard was added to each sample

extract to obtain a final concentration of approximately 2 ng Sn/mL.

3.4. Matrix recovery spiking solution

The matrix spiking solution consisted of 4BT, TBT, DBT and MBT (TBT, DBT, and MBT as the

chlorides).

Matrix spike solution was added to samples at a concentration approximately 10 times the MDL.

3.5. Retention Index Solution

The calibration mixture was used a retention index solution.

4. PROCEDURE

4.1. Sample extraction and purification

Sediment and tissue samples were extracted and purified following the analytical procedures

described in previous sections.

4.2. High resolution GC-FPD analysis

4.2.1. GC conditions

 For the analysis of butyltins, the analytical system, or its equivalent, included at a minimum:

Instrument: Hewlett-Packard 5880A or

HP 5890 Gas Chromatograph

Features: Split/splitless capillary inlet

system and data acquisition system

Inlet: Splitless

Detector: Flame photometric, 610 nm filter

Column: 0.32-mm i.d. x 30-m DB-5 fused

silica capillary column (J&W Scientific) or similar

169
Gases:

Carrier: Helium 2 mL/min.

Make-Up: Helium 33 mL/min.

Detector: Air 100 mL/min.

Hydrogen 80 mL/min.

Temperatures:

Injection port: 250 °C

Detector: 250 °C

Oven Prog: 80 °C to 300 °C at 13°/min, hold 0 min.

Run Time: 13 min.

The GC oven temperature program may be modified to improve resolution.

Daily Calibration: Mid-level calibration solution;

Retention index solution

(20 mg/mL)

Quantification: Based on surrogate added prior to extraction/calibration.

Note: The GC must be capable of the baseline resolution of all target compounds, surrogates,

and internal standards.

4.2.2. Calibration

Prior to analysis, a sample set was used to perform the GC calibration at a minimum of three

concentrations. One of the concentration levels was near, but above the MDL. The remaining

concentrations corresponded to the expected concentration range of the samples. A

concentration range of 1.25 to 50 ng Sn/μL was used. The detector was linear within this

range. An average calibration factor from the authentic standard of each individual compound

was used to calculate sample analyte concentrations. The initial calibration was verified by the

measurement of calibration standards after at least every 12 samples.

A mid-level standard was analyzed immediately prior to conducting any analyses, and after

each group of samples. The response factor criteria for an in-control calibration check was

±30% for each analyte.

4.2.3. Retention time windows

Retention time windows were established and maintained. Three times the standard deviation of

the retention time determined from the calibration solutions was used to calculate the

acceptable retention time windows.

4.2.4. Sample analysis

If the instrument was in calibration, the butyltin analyses were initiated with a calibration

check, followed by the samples, and ending with a calibration check (Table 1). If the response

factor for any analyte in the calibration check failed to meet the criteria established in Section

4.2.2, the instrument was recalibrated. All samples that were injected after the standard

exceeding the criteria were reinjected.

Sample injections of 1 to 4 μL were made with an autosampling device.

170
Table 1. Sample distribution to meet QA requirements during a typical TBT analysis.

Sample no. Description Function

1 Solvent Blank Establish instrument background

2 Cal Ck* Confirm "in calibration" condition

3 Sample #1 Unknown Analysis

4 Sample #2 Unknown Analysis

5 Sample #3 Unknown Analysis

6 Sample #4 Unknown Analysis

7 Sample #5 Unknown Analysis

8 Sample #6 Unknown Analysis

9 Sample #7 Unknown Analysis

10 Sample #8 Unknown Analysis

11 Sample #9 Unknown Analysis

12 Sample #10 (duplicate)* * Unknown Analysis

13 System Blank Confirm method blank

14 Spiked Blank/Fortified Sample/SRM Complete Analytical QA* *

15 Cal Ck Confirm "in calibration" condition

* Criteria ±30% for an individual analyte

** Criteria ±30% for all analytes

Note: The period of time in between Cal Ck did exceed 12 hours therefore the number of samples were adjusted accordingly.

If the response for any peak exceeded the highest calibration solution, the extract was diluted

and reanalyzed.

4.2.5. Calculations

Calculations were based on the internal standard methods. Concentrations were reported as μg

Sn/g.

5. QUALITY ASSURANCE/QUALITY CONTROL (QA/QC) REQUIREMENTS

5.1. Initial calibration and continuing calibration checks

Prior to the analyses, a three-point response factor linear calibration curve was established.

Each calibration standard was analyzed and the response factor (RF) of each compound was

calculated at each concentration level from the area of the peak and the Sn concentration of the

standard.

The following formula was used to calculate the response factors of the surrogate standard

relative to each of the calibration standards.

RF = 

As C su

Asu C s

171
where As

 was the area for the analyte to be measured, Asu was the area for the appropriate

surrogate standard (tripropyltin or dimethyltin), Csu was the concentration of the appropriate

surrogate standard (ng/mL), and Cs was the concentration of the analyte in the calibration

standard (ng/mL).

The response factor for each compound of interest relative to the internal standard was

determined at least once a day.

The daily response factor for each compound was compared to the initial calibration curve. The

percent difference was calculated using the following equation:

Percent difference = 

RFI - RFC

RFI 100

where RFI was the average response factor from initial calibration and RFD was the response

factor from current verification check standard.

If the average daily response factors for all analytes was within ±15% of the corresponding

calibration curve value, the analysis proceeded. If, for any individual analyte, the daily

response factor exceeded ±30% of the corresponding calibration curve value, a three-point

calibration curve was repeated prior to the analysis of the samples. All samples were

calculated based on the initial calibration response factor.

5.2. Method blank analysis

An acceptable method blank analysis did not contain any target compound at concentration 3

times greater than the MDL. If the method blank did not meet these criteria, the analytical

system was out of control and the source of the contamination was investigated, corrective

measures taken and documented before further sample analysis proceeded.

5.3. Surrogate compound analysis

All samples and quality control samples were spiked with TPT and DMT. The surrogate solutions

were spiked into the sample prior to extraction to measure individual sample matrix effects

associated with sample preparation and analysis.

The laboratory took corrective action whenever the recovery of the surrogate was below 30

percent.

The following corrective actions were taken when an out of control event occurred:

a. Calculations were checked to assure that no errors were made.

b. The internal standard and surrogate solutions were checked for degradation,

contamination, or concentration and the instrument performance was checked.

c. If the surrogate could not be measured because the sample required a dilution, no

corrective action was required. The surrogate recovery was properly annotated.

d. If the steps above failed to reveal a problem, the sample or extract was reanalyzed. If

reanalysis of the extract yielded surrogate recoveries within the stated limits, then

the reanalyzed data was reported. If upon reinjection QA criteria were still violated,

the sample was submitted for re-extraction if sufficient sample was available. If the

172
sample was completely consumed, the data was reported but designated as outside the

QA criteria.

5.4. Matrix spike analysis

The laboratory spiked and analyzed a matrix spike every 12 samples or with every sample set,

which ever was more frequent. A sample was randomly chosen, split into two subsamples and

the subsample was fortified with the matrix spike. The acceptable matrix spike recovery

criteria were 90 ± 20% for 4BT, TBT, DBT and 60 ± 25% for MBT. If the matrix spike criteria

were not met, the matrix spike was reinjected on the GC and reanalyzed. If the reinjected

matrix spike analysis met the criteria, the reanalysis data was reported. If not, the entire

batch of samples was submitted for re-extraction if sufficient sample was available. If the

sample was completely consumed, the data was reported but designated as outside the QA

criteria.

5.5. Method detection limit

The method detection limit was determined following the procedures outlined in Federal

Register (1984), Vol. 49, No. 209: 198-199.

5.6. GC resolution

The target compounds, and internal standard were resolved from one another and from

interfering compounds.

5.7. Reference sample analysis

When available, one reference material was analyzed per batch of samples, and the results used

to construct laboratory QC charts. The result should agree within ±30% of the mean of the

previously reported data on the certified concentration.

6. CALCULATIONS

6.1. Butyltin calculations

Concentrations were based on the surrogate added before extractions:

C = 

As I su

Asu RF W

where C was the sample concentration (ng Sn/g), As was the area of the analyte measured, Asu

was the area of the appropriate surrogate standard, RF is the response factor, Isu was the

amount in ng of appropriate surrogate standard added to each sample, and W is the sample dry

weight in grams.

6.2. Calculation notes

A specific amount of surrogate was added to each sample. The recovery of this surrogate was

monitored in each sample using the response of the IS. that was added to the final extract.

173
Percent SUR recovery = 

Ais C is

Csur A su RFsur

where Asu was the area of tripropyltin or dimethyltin, Ais is the area of tetrapropyltin, Csur

was the amount (in ng) of Sn in tripropyltin added to the sample, Cis was the amount (in ng) of

Sn in tetrapropyltin added to the sample extract, and RFsur was the response factor for

tripropyltin.

7. REPORTING

Report units were ng Sn/g. Results were reported to three (3) significant figures. Surrogate

recoveries were reported for every sample analyzed. Matrix spike recoveries were reported

for each batch of samples analyzed. When available, the results of the analysis of reference

materials were reported with each batch of samples analyzed.

8. CONCLUSIONS

The flame photometric detector with a 610 nm filter provides a sensitive detector that is

specific to tin. Hexylation of the organotin anions provides compounds amenable to the GC/FPD

technique and provides reliable quantitation of organotin compounds at low concentrations (ng

Sn/g).

174
Battelle Duxbury Operations Trace Organic Analytical Procedures for the

Processing and Analysis of Tissue Samples

C. S. Peven McCarthy and A. D. Uhler

Battelle Duxbury Operations

Duxbury, Massachusetts

ABSTRACT

This document describes the analytical methods for the measurement of trace organic

compounds in tissues. The methods presented are modifications of methods initially

developed by the NOAA/National Marine Fisheries Service National Analytical Facility.

These Battelle methods have been used for multiple monitoring programs, including the

analysis of samples collected for the NOAA Status and Trends Mussel Watch Project.

1. INTRODUCTION

This document summarizes the methods used by Battelle for the preparation and analysis of

tissues for trace organic contaminants. These methods are based on those developed by Sloan et

al. (1993). All equipment, apparatus, standards, and reagent necessary to perform the

extractions and analyses are described below. These methods were followed by Battelle from

1986 through 1994, as well as in 1996 and 1997 as participants in the Mussel Watch Project.

They are also the foundation for multiple monitoring and assessment projects conducted by

Battelle worldwide. The methods are continually evolving to reflect the incorporation of new

state-of-the-art analytical techniques.

2. EQUIPMENT AND REAGENTS

2.1. Sample processing equipment and apparatus

Tissue preparation equipment

Stainless steel shucking knives

Electronic calipers

500-mL certified cleaned glass sample jars (ESS, I-Chem or equivalent)

Apparatus for homogenizing tissues

Tekmar Tissumizer (Cincinnati, OH) with stainless steel probes, or equivalent

Apparatus for shaking tissue extract

Orbital shaker table; Lab-Line Instruments Inc. Model 3520 or equivalent

Apparatus for determining wet weight and dry weight (tissue and sediment)

Top-loading balance capable of weighing to 0.01 g; Ohaus, Mettler or equivalent

Aluminum weighing pan

Stainless steel spatula

Drying oven maintained at 105-120 °C, Blue M Model SW-17TA or equivalent

Apparatus for determining lipid weight:

Class A volumetric pipette/pipette bulb, or 10-mL syringe

Aluminum weighing pan

175
General sample processing equipment

250-mL Teflon or glass (certified clean) extraction bottles

Centrifuge capable of holding 250-mL bottles and maintaining 2000 rpm

Glass wool

19-mm chromatography column with ~200-mL reservoir and Teflon stopcock (tissue)

Kuderna-Danish (K-D) apparatus (listed below) or Turbovap concentration units and tubes

Reservoir, 500 mL

Snyder column, three-ball macro

Concentrator tube, 10- or 20-mL

Hot water bath capable of reaching 100 °C, located in fume hood.

Boiling chips, Teflon, solvent rinsed

Nitrogen evaporation apparatus, N-Evap, Turbovap, or equivalent

Glass graduated cylinders, 100- and 500-mL

Erlenmeyer flasks, 250- and 500-mL

Microliter syringes, Hamilton

Vials, 70-, 4-, 1-, 250-mL

Aluminum foil

Pasteur pipettes

Forceps, stainless steel

Electrobalance, Cahn, for standard preparation

High pressure liquid chromatography:

Autosampler, 231/401. Gilson

Phenogel columns, 22.5 x 250 mm, 100 Å size.

UV/VIS detector, Spectra-Physics

Fraction collector, Isco Foxy 200

Integrator, Shimadzu CR-3A

Gas chromatographs (GCs):

GCs - Hewlett Packard 5890 Series II

Mass spectrometer (MS) - Hewlett Packard 5972 MSD

Electron capture detector (ECD) - Hewlett Packard

Fused silica capillary column (30-m x 0.32 mm i.d. DB-5 MS or 0.25 mm i.d. DB-5)

Gases:

Argon/methane (95/5) (99.999% pure)

Helium (99.999% pure)

Hydrogen (99.9999% pure)

2.2. Reagents

Dichloromethane (DCM), pesticide grade or equivalent

Isooctane, pesticide grade or equivalent

Acetone, pesticide grade or equivalent

Sodium sulfate - anhydrous, reagent grade, heated to 400 °C for at least 4 hr, then cooled and

stored in a tightly-sealed glass container at room temperature.

Alumina, Sigma F-20 or equivalent, 80-200 mesh. Prepare 2% deactivated alumina by heating

to 400 °C for at least 4 hr in a shallow dish, adding 2% water (by volume) and shaking the

sealed glass flask for 1 hr. Use within 24 hr.

176
2.3. Standard solutions

Surrogate internal standards (SIS)

Recovery internal standards (RIS)

Matrix spike solutions

HPLC calibration solution

3. SUMMARY OF PROCESSING PROCEDURES

3.1. Initial extraction

Approximately 30 g of wet tissue homogenate was weighed into a Teflon extraction container

(250 mL) and spiked with surrogate internal standards (SIS). An additional 5 g aliquot of the

homogenate was placed in a pre-weighed aluminum pan for dry weight determination (see

Section 5.0). Fifty g of sodium sulfate and 75 mL DCM were added to the tissue and the mixture

homogenized for two min at high speed. The jar was centrifuged, the solvent poured off into a

labeled 500-mL Erlenmeyer flask, an additional 75 mL DCM was added to the Teflon jar and the

procedure repeated. After the second aliquot of DCM was added to the Erlenmeyer, 50 mL DCM

was added to the Teflon jar and the mixture shaken for approximately one hour. The jar was

again centrifuged and the solvent added to the other two aliquots. The Erlenmeyer was swirled

to mix the solvent extracts, and a 10-mL aliquot removed for lipid weight determination (DCM

extractables) using a Class A pipette. The aliquot was placed in a pre-weighed aluminum pan,

covered with foil, and allowed to dry at room temperature (see Section 5.0 for lipid weight

calculation). Approximately 20 - 50 g sodium sulfate was added to the Erlenmeyer and the

mixture swirled. After approximately 1 hr the combined extract was decanted through a

prepared alumina column, and the eluent collected into a clean reservoir: Erlenmeyer flask, K-D

apparatus, or Turbovap tube. When the extract reached the top of the column packing, 50 mL of

DCM is added to the column. The column was completely drained into the reservoir.

3.2. HPLC cleanup

After alumina column cleanup, the extracts are concentrated by KD and nitrogen techniques to

900 μL (volume measured with a syringe and recorded). 600 μL of this concentrated extract is

fractionated by HPLC following procedures developed by Krahn et al. (1988), and modified by

Battelle to accommodate local equipment. The system is calibrated prior to fractionation by

analyzing two calibration standards containing the retention time markers

dibromooctafluorobiphenyl (DBOFB) and perylene. The solution also contains corn oil (lipid

material) and sulfur (a common contaminant), to ensure that they will be eliminated from the

final cleaned extracts. The fractionation window is set to collect between 1.5 min before the

elution of DBOFB until 2 min after the elution of perylene. The pump is set to flow at a rate of 5

mL/min (DCM mobile phase). A calibration check is run every 10 samples to verify the

collection window.

After the samples were fractionated, the cleaned extracts were concentrated under nitrogen to

approximately 0.5 mL. The extracts were spiked with internal standards and split for GC and

GC/MS analysis. The GC/MS split was transferred to a GC vial and submitted for polycyclic

aromatic hydrocarbons (PAH) analysis; the GC split was solvent-exchanged with isooctane,

transferred to a GC vial, and submitted to GC/ECD for polychlorinated biphenyl (PCB)/pesticide

analysis.

177
4. SUMMARY OF ANALYSIS METHODS

4.1. Calibration

Prior to sample analysis, the gas chromatography system was calibrated using stock standard

solutions containing the analytes of interest as well as the appropriate surrogates and internal

standards. Five-level calibrations are analyzed by both GC/ECD (for PCB and pesticides) and

GC/MS operating in the SIM mode (for PAH). The system was checked prior to each sample set,

and the calibration checked every 10 samples (minimum) by analyzing one of the calibration

levels and checking target analyte response. The analyte response factor must fall within 25%

of the response factor from the initial calibration for the calibrations to be considered

acceptable.

4.2. GC/ECD

The GC is fitted with a 30-m x 0.25-mm internal diameter fused silica capillary column with a

0.25-μm film thickness stationary phase [DB-5 (5% phenyl-, 95% methyl-polysiloxane) (J&W

Scientific, Inc.)]. GC conditions are as follows:

Injection port temperature 280 °C

Detector temperature 300 °C

Initial temperature 60 °C

Initial hold 1 min

Ramp 1 rate 15 °C/min to 150 °C

Ramp 2 rate 1 °C/min to 210 °C

Ramp 3 rate 10 °C/min to 290 °C

Final hold 15 min

Carrier gas flow 1.5 - 2.5 mL/min (30 - 40 cm/sec)

Makeup gas flow 30 - 60 mL/min

Purge vent 1.5 - 2.5 mL/min

Split vent 40 - 80 mL/min

Purge on after 0.50 min

One or 2 μL injections of the sample extracts are made onto the column. Column degradation

checks are made prior to each sample sequence. The degradation solution contains endrin and

4,4’-DDT.

4.3. GC/MS

Polynuclear aromatic hydrocarbons were separated via high resolution capillary gas

chromatography, and identified and quantified using electron impact mass spectrometry. A

Hewlett Packard system interfaced to the GC/MS is used to control acquisition and to store,

retrieve, and manipulate mass spectral data.

Prior to the analysis of analytical standards and/or samples the mass spectrometer must be

tuned with perfluorotributlyamine (PFTBA).

The gas chromatograph is fitted with a 30-m x 0.25-mm DB-5MS (J&W Scientific, Inc.) or

equivalent (0.25-μm film thickness) fused silica capillary column. The GC oven temperature

program is as follows:

Injection Port: 300 °C

Transfer Liner: 280 °C

Initial Temp: 40 °C

178
Initial Hold: 1 min

Ramp Rate: 6 °C/min

Final Temp: 290 °C

Final Hold: 20 min

A 2 μL injection volume of sample extract was used for analysis. The mass spectrometer is

operated in electron impact mode at a manifold pressure of less than 6 x 10-5 torr. The

electron multiplier voltage was set a minimum of 100 V above the tune value.

The quantification and confirmation ions used in the analysis of selected PAH were defined in

Battelle standard operating procedures. If possible, ion groups should be selected so that no

more than 20 ions are monitored in a single group. It should be noted that as the number of ions

scanned per group increases and the individual dwell times decrease, sensitivity will also

decrease. Each ion in a group should have identical dwell times to ensure that correct ion ratios

are preserved. Total group dwell time should not exceed 400 ms, individual dwell times should

be a minimum of 20 ms.

Prior to the first analysis of analytical standards and/or samples in SIM, a 10 ng/μL analytical

standard and reference sample (e.g. North Slope Crude Oil) is analyzed in full-scan. The fullscan total ion chromatograms from these analyses are used to determine the proper group start

and stop times in the SIM acquisition method.

5. CALCULATIONS

Sample dry weight:

% Dry weight = 

(Aliquot dry wt. - pan wt.)

(Aliquot wet wt. - pan wt.) x 100

Lipid weight:

Sample dry wt. = 

(% dry wt.) (sample wet wt.)

(100)

Total lipid wt. (g) = 

Vol. of combined extracts (mL)

Aliquot vol. (mL) x (Aliquot dry wt. (g) - Tare wt. (g))

Sample lipid content (g/g dry wt.) = 

Total lipid wt. (g)

Sample dry wt. (g)

Calibration response factor:

RF = 

Area of analyte

Area of Internal Standard x 

Concentration of Internal Standard

Concentration of analyte

6. CONCLUSION

The method described above has been validated and is continually being reviewed and modified

to incorporate improvements in order to keep it current and efficient. The methods are the

basis for all tissue monitoring studies where ultra-low detection limits of NS&T and similar

organic contaminants are required.

179
7. REFERENCES

Krahn, M. M., C. A. Wigren, R. W. Pearch, L. K. Moore, R. G. Bogar, W. D. MacLeod, Jr., S. L.

Chan, and D. W. Brown (1988) A rapid high-performance liquid chromatographic method for

isolating organic contaminants from tissue and sediment extracts. J. Chromatogr., 437:167-

75.

Sloan, C. A., N. G. Adams, R. W. Pearce, D. W. Brown, and S. L. Chan (1993) Northwest

Fisheries Science Center organic analytical procedures. In: Sampling and Analytical Methods of

the National Status and Trends Program: National Benthic Surveillance and Mussel Watch

Programs: 1984-1992. G. G. Lauenstein and A. Y. Cantillo (eds.) NOAA Technical Memorandum

NOS ORCA 71. NOAA/NOS/ORCA, Silver Spring, MD. pp. IV.35-IV.97.

180
181
NIST Methods for the Certification of SRM 1941a and SRM 1974a

M. M. Schantz, B. A. Benner, Jr., M. K. Donais,* M. J. Hays,* * W. R. Kelly, R. M. Parris, B. J.

Porter, D. L. Poster, L. C. Sander, K. S. Sharpless, R. D. Vocke, Jr., and S. A. Wise

Analytical Chemistry Division

National Institute of Standards and Technology

Gaithersburg, MD

M. Levenson, S. B. Schiller, and M. Vangel

Statistical Engineering Division

National Institute of Standards and Technology

Gaithersburg, MD

ABSTRACT

The replacement Standard Reference Materials (SRMs) for SRM 1941, Organics in

Marine Sediment, and SRM 1974, Organics in Mussel Tissue (Mytilus edulis), have been

prepared and analyzed for the determination of trace organic constituents. SRM 1941a

has been issued with certified concentrations for 23 polycyclic aromatic hydrocarbons

(PAH), 21 polychlorinated biphenyl (PCB) congeners, 6 chlorinated pesticides, and

sulfur. SRM 1974a has been issued with certified concentrations for 15 PAHs, 20 PCB

congeners, 7 chlorinated pesticides, and methylmercury. The preparation and

certification analyses for these two replacement SRMs are described in this chapter.

1. INTRODUCTION

As part of the Quality Assurance element of the National Status and Trends Program, the

National Institute of Standards and Technology (NIST) developed two Standard Reference

Materials (SRMs), SRM 1941 (Organics in Marine Sediment) and SRM 1974 [Organics in Mussel

Tissue (Mytilus edulis)], to assist in the validation of analytical procedures. SRM 1941 was

issued in 1989 with certified concentrations for 11 polycyclic aromatic hydrocarbons (PAHs)

and sulfur and noncertified concentrations for polychlorinated biphenyl (PCB) congeners and

chlorinated pesticides (Schantz et al., 1990). SRM 1974 was issued in 1990 with certified

concentrations for nine PAHs and noncertified concentrations for PCB congeners and chlorinated

pesticides (Wise et al., 1991). The supplies of these original SRMs were depleted in 1993 (SRM

1941) and 1994 (SRM 1974) so new materials, SRM 1941a and SRM 1974a, have been

prepared and analyzed. To increase the usefulness of the new SRMs, three goals were

established: (1) prepare a larger quantity of the material to extend the availability, (2) provide

certified values for a significantly larger number of PAHs and to reduce the uncertainties

associated with the certified PAH mass fractions compared to the original SRMs, and (3)

provide certified values for the PCB congeners and chlorinated pesticides rather than

noncertified values as in the original materials (Schantz et al., 1995).

At NIST, the certification process for a natural matrix SRM typically requires the use of two

or more chemically independent analytical techniques, and the results of these analyses, if in

agreement, are used to determine the certified values of the measured analytes. For the

measurement of PAHs, Wise et al. (1995) described the use of four different analytical

techniques: (1) reversed-phase liquid chromatography with fluorescence detection (LC-Fl) for

analysis of the total PAH fraction, (2) reversed-phase LC-Fl analysis of isomeric PAH

*

 Present address: VG Elemental, 27 Forge Parkway, Franklin, MA 02038.

** Present address: Battelle Columbus, 505 King Ave., Columbus, OH 43201.

181
fractions isolated by normal-phase LC (i.e., multidimensional LC), (3) gas

chromatography/mass spectrometry (GC/MS) analysis of the PAH fraction on a 5% phenylsubstituted methylpolysiloxane stationary phase, and (4) GC/MS of the PAH fraction on a

smectic liquid crystalline stationary phase that provides excellent shape selectivity for the

separation of PAH isomers. Based on the agreement of results from these four approaches,

certified values were determined for 23 PAHs in SRM 1941a and 15 PAHs in SRM 1974a while

noncertified values were reported for an additional 14 PAHs in SRM 1941a and 18 PAHs in SRM

1974a.

To provide certified concentrations for the PCB congeners and chlorinated pesticides, the

approaches used for SRM 1941a and SRM 1974a were similar to that used for SRM 1945,

Organics in Whale Blubber (Schantz et al., 1995). This approach consists of GC analyses with

electron capture detection (GC-ECD) on a 5% phenyl-substituted methylpolysiloxane phase and

on a dimethylpolysiloxane phase containing 50% methyl C18 and GC/MS analyses on a 5%

phenyl-substituted methylpolysiloxane phase. Using this approach, certified values were

provided for 21 PCB congeners and six chlorinated pesticides in SRM 1941a and for 20 PCB

congeners and 7 chlorinated pesticides in SRM 1974a. Noncertified values were provided for 10

additional PCB congeners/chlorinated pesticides in SRM 1941a and for eight additional PCB

congeners/chlorinated pesticides in SRM 1974a.

Additional values included for SRM 1941a are a certified value for sulfur and noncertified

values for 27 inorganic constituents and percent total organic carbon (TOC). Additional values

included for SRM 1974a are a certified value for methylmercury (Donais et al., 1996) and

noncertified values for 28 inorganic constituents.

2. SRM 1941a, ORGANICS IN MARINE SEDIMENT

2.1. Summary

SRM 1941a is intended for use in validating analytical methods for the determination of trace

levels of selected PAHs, PCB congeners, chlorinated pesticides, and sulfur in marine sediments.

Noncertified values of additional PAHs, PCB congeners, chlorinated pesticides, and inorganic

constituents are also provided.

2.2. Collection and preparation

The marine sediment used to prepare this SRM was collected in the Chesapeake Bay at the

mouth of the Baltimore (MD) Harbor near the Francis Scott Key Bridge using a modified Van

Veen type grab sampler designed to sample to a depth of 10 cm. The sediment was freezedried, sieved (150-250 μm particles used for the SRM), homogenized in a cone blender,

radiation (60Co) sterilized, and then packaged in screw-capped amber glass bottles (50

g/bottle).

2.3. Moisture determination

The results for both the organic and inorganic constituents are reported on a dry weight basis;

however, the material as received contains residual moisture. The amount of moisture in SRM

1941a was determined by measuring the weight loss after oven drying at 90 °C for 18 hr for

subsamples of 1-2 g. The moisture content in SRM 1941a at the time of the certification

analyses was 2.2%.

182
2.4. Polycyclic aromatic hydrocarbons

SRM 1941a was analyzed for selected PAHs using GC/MS and reversed-phase LC-Fl. GC/MS

analyses were performed on two columns with different selectivities for the separation of

PAHs: a 5% phenyl-substituted methylpolysiloxane stationary phase and a smectic liquid

crystalline stationary phase.

For the GC/MS analyses, two sets of six samples (10-25 g each) from twelve randomly

selected bottles were Soxhlet extracted for 18 - 20 hr using dichloromethane. A silica or

aminopropylsilane solid phase extraction column was eluted with 1:50 (volume ratio)

dichloromethane in n-hexane to remove the polar interferences from each sediment extract.

Finely divided copper was then added to the extracts to remove elemental sulfur. The PAH

fraction was isolated from each sediment extract by normal-phase liquid chromatography using

a semi-preparative aminopropylsilane column (Wise et al., 1977). The GC/MS analyses were

performed on one set of six sample extracts using a 0.25 mm i.d. x 60 m fused silica capillary

column with a 5% phenyl-substituted polysiloxane phase (0.25 μm film thickness) (DB-5MS,

J&W Scientific, Folsom, CA). The second set of six sample extracts was prepared as described

above and then analyzed by GC/MS on a 0.2 mm i.d. x 25 m fused silica capillary column with a

smectic liquid crystalline phase (0.15 μm film thickness) (SB-Smectic, Dionex, Inc., Salt Lake

City, UT).

For the LC-Fl analyses, subsamples of 10 g of sediment from six randomly selected bottles

were Soxhlet extracted for 20 hr using 1:1 (volume ratio) n-hexane:acetone. Each

concentrated extract was placed on an aminopropylsilane solid phase extraction column and

eluted with 1:50 (volume ratio) dichloromethane in n-hexane to remove the polar constituents.

The extract was then analyzed by reversed-phase LC using a polymeric octadecylsilane (C18)

column (4.6 mm i.d. x 25 cm, 5 μm particle size, Hypersil PAH, Keystone Scientific, Inc.

Bellefonte, PA) with wavelength programmed fluorescence detection (May and Wise, 1984;

Kline et al., 1985; Wise et al., 1988). To quantify several PAHs that have low fluorescence

sensitivity and /or selectivity, six additional subsamples were extracted and prepared as

described above, and then the extract was fractionated on a semi-preparative

aminopropylsilane column to isolate isomeric PAH fractions as described previously (May and

Wise, 1984; Kline et al., 1985; Wise et al., 1988). These isomeric PAH fractions were

analyzed by reversed-phase LC-Fl on a similar octadecylsilane column.

For both the GC/MS and LC-Fl analyses, selected perdeuterated PAHs were added to the

sediment prior to extraction for use as the internal standards for quantification purposes. For

GC/MS analyses the following internal standards were used: naphthalene-d8, acenaphthened10, fluorene-d10, phenanthrene-d10, fluoranthene-d10, benz[a]anthracene-d12,

benzo[e]pyrene-d12, dibenz[a,h]anthracene-d14, and benzo[ghi]perylene-d12. For the LC-Fl

analyses the following internal standards were used: naphthalene-d8, phenanthrene-d10,

fluoranthene-d10, and perylene-d12 for the LC-Fl (Total) analyses and triphenylene-d12,

benz[a]anthracene-d12, and benzo[ghi]perylene-d14 for the LC-Fl (Fraction) analyses.

Calibration response factors for the analytes relative to the internal standards were

determined by analyzing aliquot of SRMs 1491 (Aromatic Hydrocarbons in Hexane/ Toluene)

and 2260 (Aromatic Hydrocarbons in Toluene, Nominal Concentration 60 μg/mL) in the case of

GC/MS analyses or SRM 1647b (Priority Pollutant PAHs in Acetonitrile) in the case of LC-Fl

analyses; gravimetrically prepared solutions of additional analytes not contained in SRMs

1491, 2260, or 1647b; and the internal standards.

183
2.5. Polychlorinated biphenyl congeners and chlorinated pesticides

SRM 1941a was analyzed for selected PCB congeners and chlorinated pesticides using GC-ECD

and GC/MS using the general approach described previously (Schantz et al., 1993). For the GCECD analyses, a portion of the extract prepared for the PAH analyses by GC/MS (see above)

was used. For the GC/MS analyses, additional samples of approximately 100 g each form six

pairs of randomly selected bottles were Soxhlet extracted for 18 hr using 1:1 (volume ratio)

n-hexane:acetone. In preparation for both the GC-ECD and GC/MS analyses, the extracts were

fractionated on the semi-preparative aminopropylsilane column to isolate two fractions

containing (1) the PCBs and lower polarity pesticides and (2) the more polar pesticides. GC-ECD

analyses of the two fractions were performed on two columns of different selectivities for PCB

separations: 0.25 mm x 60 m fused silica capillary column with a 5% phenyl-substituted

methylpolysiloxane phase containing 50% methyl C18 (0.1 μm film thickness) (CP Sil 5 C18 CB,

Chrompack International, Middelburg, The Netherlands). The GC/MS analyses were performed

on a 5% phenyl-substituted methylpolysiloxane phase as described above for the GC-ECD

analyses.

Two PCB congeners, which are not present at significant concentrations in the sediment extract

(PCB 103 and PCB 198) (Ballschmiter and Zell, 1980; Schulte and Malisch, 1983), and

perdeuterated 4,4'-DDT were added to the sediment prior to extraction for use as internal

standards for quantification purposes. Calibration response factors for the analytes relative to

the internal standards were determined by analyzing aliquot of SRMs 2261 (Chlorinated

Pesticides in Hexane, Nominal Concentration 2 μg/mL), SRM 2262 (Chlorinated Biphenyl

Congeners in 2,2,4-Trimethylpentane, Nominal Concentration 2 μg/mL), gravimetrically

prepared solutions of additional analytes not contained in SRMs 2261 and 2262, and the internal

standards.

2.6. Certified and noncertified concentrations

Statistically combining the results from the different methods, SRM 1941a has been issued

with certified values for 23 PAHs (Table 1), 21 PCB congeners (Table 2), and 6 chlorinated

pesticides (Table 3). Noncertified values are available for an additional 14 PAHs (Table 4), 7

PCB congeners (Table 5), and three chlorinated pesticides (Table 5). In addition to the organic

contaminants, the sulfur content was determined to be 0.9589 ± 0.0058% (by weight) using

isotope dilution thermal ionization MS and the total organic carbon was determined to be 4.8 ±

1.2% (by weight) from an interlaboratory comparison. Also, SRM 1941a was analyzed for 27

inorganic constituents using instrumental neutron activation analysis (Wise et al., 1995).

3. SRM 1974a, ORGANICS IN MUSSEL TISSUE (Mytilus edulis)

3.1. Summary

SRM 1974a is intended for use in validating analytical methods for the determination of trace

levels of selected PAHs, PCB congeners, chlorinated pesticides, and methylmercury in mussel

tissues. Noncertified values of additional PAHs, PCB congeners, chlorinated pesticides, and

inorganic constituents are also provided.

184
Table 1. Certified concentrations (mass fractions) for selected PAHs in SRM 1941a and SRM

1974aa (μg/kg dry mass basisb).

Compound SRM 1941a SRM 1974a

Naphthalene 1010 ± 140 23.5 ± 4.4c

Fluorene 97.3 ± 8.6

Phenanthrene 489 ± 23 22.2 ± 2.4

Anthracene 184 ± 14 6.1 ± 1.7c

Fluoranthene 981 ± 78 163.7 ± 9.1c

Pyrene 811 ± 24 151.6 ± 6.6c

Benz[a]anthracene 427 ± 25 32.5 ± 4.7c

Chrysene 380 ± 24 44.2 ± 2.3

Triphenylene 197 ± 11 50.7 ± 5.9

Benzo[b]fluoranthene 740 ± 110 46.4 ± 3.7c

Benzo[k]fluoranthene 361 ± 18 20.18 ± 0.84

Benzo[a]fluoranthene 118 ± 11

Benzo[e]pyrene 553 ± 59 84.0 ± 1.9

Benzo[a]pyrene 628 ± 52 15.63 ± 0.65

Perylene 452 ± 58 7.68 ± 0.27

Benzo[ghi]perylene 501 ± 72 22.0 ± 2.2c

Indeno[1,2,3-cd]pyrene 525 ± 67 14.2 ± 2.8c

Dibenz[a,j]anthracene 74.3 ± 6.8

Dibenz[a,c]anthracene 43.1 ± 3.7

Dibenz[a,h]anthracene 73.9 ± 9.7

Pentaphene 42 ± 12

Benzo[b]chrysene 99 ± 20

Picene 80.0 ± 9.0

a Results reported on dry mass basis. SRM 1941a as received contains approximately 2.2% moisture, and SRM 1974a as

received contains 88.61% ± 0.08% water.

b Each certified value is the mean of the equally weighted means from two or more independent analytical methods. Each

uncertainty, computed according to the CIPM approach (Guide, 1993), is an expanded uncertainty at the 95% level of

confidence that includes random sources of uncertainty within each analytical method as well as uncertainty due to the

drying study (for the dry mass basis values). The expanded uncertainty defines a range of values for the certified value

within which the true value is believed to lie, at a level of confidence of approximately 95%.

c An additional allowance for differences between methods was included in the total uncertainty for this analyte.

185
Table 2. Certified concentrations (mass fractions) for selected PCB congeners in SRM 1941a

and SRM 1974aa (μg/kg dry mass basisc).

Chlorinated biphenylsb SRM 1941a SRM 1974a

PCB 44 (2,2',3,5'-Tetrachlorobiphenyl) 4.80 ± 0.62 72.7 ± 7.4

PCB 49 (2,2',4,5'-Tetrachlorobiphenyl) 9.5 ± 2.1 88.8 ± 5.0

PCB 52 (2,2',5,5'-Tetrachlorobiphenyl) 6.89 ± 0.56 115 ± 11

PCB 66 (2,3',4,4'-Tetrachlorobiphenyl) 6.8 ± 1.4 101.4 ± 4.4

PCB 87 (2,2',3,4,5'-Pentachlorobiphenyl) 6.70 ± 0.37

PCB 95 (2,2',3,5',6-Pentachlorobiphenyl) 7.5 ± 1.1 83 ± 17

PCB 99 (2,2',4,4',5-Pentachlorobiphenyl) 4.17 ± 0.51 70.9 ± 4.0

PCB 101 (2,2',4,5,5'-Pentachlorobiphenyl) 11.0 ± 1.6 128.3 ± 9.7

PCB 105 (2,3,3',4,4'-Pentachlorobiphenyl) 3.65 ± 0.27 53.0 ± 3.4

PCB 110 (2,3,3',4',6-Pentachlorobiphenyl) 9.47 ± 0.85 127.3 ± 8.6

PCB 118 (2,3',4,4',5-Pentachlorobiphenyl) 10.0 ± 1.1 130.8 ± 3.6

PCB 128 (2,2',3,3',4,4'-Hexachlorobiphenyl) 1.87 ± 0.32 22.0 ± 3.4

PCB 138 (2,2',3,4,4',5'-Hexachlorobiphenyl) 13.38 ± 0.97 133.5 ± 9.5

163 (2,3,3',4',5,6-Hexachlorobiphenyl)

164 (2,3,3',4',5',6-Hexachlorobiphenyl)

PCB 149 (2,2',3,4',5',6-Hexachlorobiphenyl) 9.2 ± 1.1 87.6 ± 2.3

PCB 151 (2,2',3,5,5',6-Hexachlorobiphenyl) 25.6 ± 3.5

PCB 153 (2,2',4,4',5,5'-Hexachlorobiphenyl) 17.6 ± 1.9 145.2 ± 7.6

PCB 156 (2,3,3',4,4',5-Hexachlorobiphenyl) 0.93 ± 0.14 7.43 ± 0.99

PCB 170 (2,2',3,3',4,4',5-Heptachlorobiphenyl) 3.00 ± 0.46 5.5 ± 1.1

PCB 180 (2,2',3,4,4',5,5'-Heptachlorobiphenyl) 5.83 ± 0.058 17.1 ± 3.8

PCB 183 (2,2',3,4,4',5',6-Heptachlorobiphenyl) 16.0 ± 2.4

PCB 187 (2,2',3,4',5,5',6-Heptachlorobiphenyl) 34.0 ± 2.3

159 (2,3,3',4,5,5'-Hexachlorobiphenyl)

182 (2,2',3',4,4',5,6'-Heptachlorobiphenyl)

PCB 194 (2,2',3,3',4,4',5,5'-Octachlorobiphenyl) 1.78 ± 0.23

PCB 206 (2,2',3,3',4,4',5,5',6-Nonachlorobiphenyl) 3.67 ± 0.87

PCB 209 (Decachlorobiphenyl) 8.34 ± 0.49

a Results reported on dry mass basis. SRM 1941a as received contains approximately 2.2% moisture, and SRM 1974a as

received contains 88.61% ± 0.08% water.

b PCB congeners are numbered according to the scheme proposed by Ballschmiter and Zell (1980) and later revised by Schulte

and Malisch (1983) to conform with IUPAC rules; for the specific congeners mentioned in this SRM, the Ballschmiter-Zell

numbers correspond to those of Schulte and Malisch. When two or more congeners are known to coelute under the conditions

used, the PCB congener listed first is the major component and the additional congeners may be present as minor

components. The quantitative results are based on the response of the congener listed first.

c Each certified value is the mean of the equally weighted means from four to seven different analytical methods. Each

uncertainty, computed according to the CIPM approach (Guide, 1993), is an expanded uncertainty at the 95% level of

confidence that incorporates within- and between-method uncertainty as well as uncertainty due to the drying study (for the

dry mass basis values). The expanded uncertainty defines a range of values for the certified value within which the true

value is believed to lie, at a level of confidence of approximately 95%.

186
Table 3. Certified concentrations (mass fractions) for selected chlorinated pesticides in SRM

1941a and SRM 1974aa (μg/kg dry mass basisb).

Chlorinated pesticides SRM 1941a SRM 1974a

Hexachlorobenzene 70 ± 25

cis-Chlordane ( -Chlordane) 2.33 ± 0.56 17.2 ± 2.8

trans-Chlordane 16.6 ± 1.7

cis-Nonachlor 6.84 ± 0.90

trans-Nonachlor 1.26 ± 0.13 18.0 ± 3.6

2,4'-DDE 0.73 ± 0.11

4,4'-DDE 6.59 ± 0.56 51.2 ± 5.5

4,4'-DDD 5.06 ± 0.58 43.0 ± 6.3

4,4'-DDT 3.91 ± 0.59

a Results reported on dry mass basis. SRM 1941a as received contains approximately 2.2% moisture, and SRM 1974a as

received contains 88.61% ± 0.08% water.

b Each certified value is the mean of the equally weighted means from four to seven different analytical methods. Each

uncertainty, computed according to the CIPM approach (Guide,1993), is an expanded uncertainty at the 95% level of

confidence that incorporates within- and between-method uncertainty as well as uncertainty due to the drying study (for the

dry mass basis values). The expanded uncertainty defines a range of values for the certified value within which the true

value is believed to lie, at a level of confidence of approximately 95%.

3.2. Collection and preparation

The mussels (Mytilus edulis) used for the preparation of SRM 1974a were collected on October

7, 1992 from Dorchester Bay within Boston Harbor, MA following the same procedures as

described previously for the collection of mussels for SRM 1974 (Wise et al., 1991).

Approximately 6000 individual mussels were collected by hand at low tide. The samples were

transported to the Battelle Ocean Sciences Laboratory (Duxbury, MA) where the mussels were

rinsed with water to remove rocks and other debris. The samples were bagged in Ziploc bags

and placed in insulated, Teflon-lined wooden containers, frozen and transported to NIST on dry

ice. The samples were transferred to Teflon bags and stored in a liquid nitrogen vapor freezer

(-120 °C) until they were shucked.

The mussel tissue was removed from the shell using the following procedure. The mussels were

allowed to warm up to about 0 °C; the tissue was removed from the shell using a titanium knife

and placed in Teflon bags (approximately 1 kg per bag) and immediately returned to a liquid

nitrogen vapor freezer. Approximately 81 kg of mussel tissue were prepared for use as the

SRM. Approximately 20 kg of the same frozen mussel homogenate were freeze-dried. The

freeze-dried material will be issued as a separate material, SRM 2974.

The mussel tissue was removed from the shell using the following procedure. The mussels were

allowed to warm up to about 0 °C; the tissue was removed from the shell using a titanium knife

and placed in Teflon bags (approximately 1 kg per bag) and immediately returned to a liquid

nitrogen vapor freezer. Approximately 81 kg of mussel tissue were prepared for use as the

SRM. Approximately 20 kg of the same frozen mussel homogenate were freeze-dried. The

freeze-dried material will be issued as a separate material, SRM 2974.

187
Table 4. Noncertified concentrations (mass fractions) for selected PAHs in SRM 1941a and SRM

1974aa (μg/kg dry mass basisb).

Compound SRM 1941a SRM 1974a

1-Methylnaphthalene 5.3 ± 1.8

2-Methylnaphthalene 10.2 ± 1.5

Biphenyl 175 ± 18 5.11 ± 0.33

Acenaphthylene 37 ± 14 5.25 ± 0.38

Acenaphthene 41 ± 10 3.15 ± 0.26

Fluorene 5.72 ± 0.91

Dibenzothiophene 70.0 ± 9.4

1-Methylphenanthrene 101 ± 27 10.5 ± 4.8

2-Methylphenanthrene 158 ± 32 20.6 ± 8.0

3-Methylphenanthrene 97 ± 32 13.5 ± 9.7

4-Methylphenanthrene/9-Methylphenanthrene 14.7 ± 9.2

4H-Cyclopenta[def]phenanthrene 92 ± 15

Acephenanthrylene 48.1 ± 1.2

Benzo[c]phenanthrene 80 ± 39 19.5 ± 6.7

Benzo[ghi]fluoranthene 97.9 ± 3.1 28.3 ± 5.5

Benzo[a]fluoranthene 4.0 ± 1.9

Benzo[j ]fluoranthene 341 ± 22 20.5 ± 1.7

Dibenz[a,j]anthracene 1.247 ± 0.075

Dibenz[a,h]anthracene/Dibenz[a,c]anthracene 3.00 ± 0.20

Benzo[b]chrysene 1.60 ± 0.15

Indeno[1,2,3-cd]fluoranthene 20.0 ± 2.3

Anthanthrene 129 ± 10 1.15 ± 0.31

a Results reported on dry weight basis. SRM 1941a as received contains approximately 2.2% moisture, and SRM 1974a as

received contains 88.61% ± 0.08% water.

b Each noncertified value is the mean of the equally weighted means from two different analytical methods or the mean

determined by one analytical method. Each uncertainty, computed according to the CIPM approach (Guide, 1993), is an

expanded uncertainty at the 95% level of confidence that includes random sources of uncertainty within each analytical

method as well as uncertainty due to the drying study (for the dry mass basis values). The expanded uncertainty defines a

range of values for the certified value within which the true value is believed to lie, at a level of confidence of approximately

95%.

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Table 5. Noncertified concentrations (mass fractions) for selected PCB congeners and

chlorinated pesticides in SRM 1941a and SRM 1974aa (μg/kg dry mass basisc).

Chlorinated biphenylsb SRM 1941a SRM 1974a

PCB 8 (2,4'-Dichlorobiphenyl) 1.39 ± 0.19

PCB 18 (2,2',5-Trichlorobiphenyl) 1.15 ± 0.16 33 ± 11

PCB 28 (2,4,4'-Trichlorobiphenyl) 9.8 ± 3.7 79 ± 15

PCB 31 (2,4',5-Trichlorobiphenyl) 6.2 ± 2.4 76 ± 21

PCB 87 (2,2',3,4,5'-Pentachlorobiphenyl) 54 ± 14

PCB 151 (2,2',3,5,5',6-Hexachlorobiphenyl) 2.62 ± 0.22

PCB 183 (2,2',3,4,4',5',6-Heptachlorobiphenyl) 1.63 ± 0.15

PCB 187 (2,2',3,4',5,5',6-Heptachlorobiphenyl) 7.0 ± 2.6

182 (2,2',3,4,4',5,6'-Heptachlorobiphenyl)

Chlorinated pesticides

Oxychlordane 2.59 ± 0.19

Dieldrin 1.26 ± 0.37 6.2 ± 1.3

2,4'-DDE 5.26 ± 0.27

2,4'-DDD 13.7 ± 2.8

2,4'-DDT 8.5 ± 1.9

4,4'-DDT 1.25 ± 0.10

a Results reported on both wet and dry mass basis; the sample as received contains 88.61% ± 0.08% water.

b PCB congeners are numbered according to the scheme proposed by Ballschmiter and Zell (1980) and later revised by Schulte

and Malisch (1983) to conform with IUPAC rules; for the specific congeners mentioned in this SRM, the Ballschmiter-Zell

numbers correspond to those of Schulte and Malisch.

c Each noncertified value is the mean of the equally weighted means from four to seven different analytical methods . Each

uncertainty, computed according to the CIPM approach (Guide, 1993), is an expanded uncertainty at the 95% level of

confidence that incorporates within- and between-method uncertainty as well as uncertainty due to the drying study (for the

dry mass basis values). The expanded uncertainty defines a range of values for the certified value within which the true

value is believed to lie, at a level of confidence of approximately 95%.

The frozen mussel tissue was pulverized in batches of approximately 700 g each using a

cryogenic procedure described previously (Zeisler et al., 1983). The pulverized material was

then homogenized in an aluminum mixing drum in 30 kg batches. The mixing drum was designed

to fit inside the liquid nitrogen vapor freezer and to rotate in the freezer thereby mixing the

frozen tissue powder. After mixing for 2 hr, subsamples of approximately 15 g of the tissue

homogenate were aliquoted into pre-cleaned, pre-cooled glass bottles fitted with screw caps

containing Teflon liners.

3.3. Moisture determination

The moisture content of SRM 1974a was determined by measuring the mass loss from freezedrying. Twenty bottles of SRM 1974a were selected according to a stratified randomization

scheme for the drying study. The entire contents of each bottle were transferred to a Teflon

bottle and dried for 5 days at 1 Pa with a -20 °C shelf temperature and a -50 °C condenser

temperature. The shelf temperature was gradually increased to 5 °C. The sample was

considered dry when a stable mass was obtained. Based on these studies, a 95% confidence

189
interval for the mean moisture content of SRM 1974a is 88.61% ± 0.08%. Analytical results

for the organic constituents were determined on a wet basis and then converted to a dry basis

by dividing by the conversion factor of 0.1139 kg dry mass/kg wet mass.

3.4. Polycyclic aromatic hydrocarbons

The general approach used for the determination of PAHs in SRM 1974a is similar to that

discussed above for the certification of SRM 1941a. Three sets of GC/MS results, designated

as GC/MS (I), GC/MS (II), and GC/MS (Sm), were obtained using two columns with different

selectivities for the separation of PAHs. For GC/MS (I) analyses, duplicate subsamples of 7 g

to 9 g (wet basis) of the mussel homogenate from 12 randomly selected bottles were mixed

with approximately 100 g of sodium sulfate, an internal standard (see below) was added to the

sodium sulfate-tissue mixture, and then the mixture was Soxhlet extracted for 18 hr using

250 mL of dichloromethane. The extract was concentrated, and size exclusion chromatography

on a semi-preparative divinylbenzene-polystyrene column (10 μm particle size, 100 Å pore

size, 2.5 cm i.d. x 60 cm) was used to remove the majority of the lipid and biogenic materials.

The eluent was concentrated and injected onto a semi-preparative aminopropylsilane column to

isolate the PAH fraction by normal-phase LC. The PAH fraction was then analyzed by GC/MS

using a 0.25 mm x 60 m fused silica capillary column with a 5% phenyl-substituted

methylpolysiloxane phase (0.25 μm film thickness, DB-5 MS, J&W Scientific, Folsom, CA).

For the GC/MS (II) analyses, subsamples of 14 g to 16 g (wet basis) from three bottles of SRM

1974a were extracted and one half of each extract was analyzed using the same procedure as

described above for GC/MS (I). However, these extractions and analyses were performed as

part of three different sample sets at different times using different calibrations, method

blanks, and quality control samples for each set.

The GC/MS (Sm) results were obtained by analyzing seven of the sample extracts from the

GC/MS (I) set using a 0.2 mm i.d. x 25 m (0.15 μm film thickness) smectic liquid crystalline

stationary phase (SB-Smectic, Dionex, Lee Scientific Division, Salt Lake City, UT). The liquid

crystalline phase provides significantly different selectivity based on molecular shape for the

separation of PAH isomers when compared with the 5% phenyl methylpolysiloxane phase. The

column has a limited temperature range.

Two sets of LC-Fl results, designated as LC-Fl (Total) and LC-Fl (Fraction), were used in the

certification process. For the LC-Fl (Total) analyses, a subsample of approximately 15 g (wet

basis) of the mussel homogenate from eight randomly selected bottles was mixed with

approximately 100 g of sodium sulfate, an internal standard (see below) was added to the

sodium sulfate-tissue mixture, and then the mixture was Soxhlet extracted for 20 hr using

200 mL of 1:1 (volume ratio) hexane:acetone. The extract was concentrated and then passed

through an aminopropylsilane solid phase extraction (SPE) column using 1:50 (volume ratio)

dichloromethane in hexane to remove the lipid and more polar interferences. The eluent from

the SPE column was concentrated and the SPE procedure was repeated five times on different

SPE columns. The cleaned up extract was then analyzed by reversed-phase LC using a

polymeric octadecylsilane (C18) column (4.6 mm i.d. x 15 cm, 3 μm particle size, ChromSpher

PAH, Chrompack, Middelburg, The Netherlands) with wavelength programmed fluorescence

detection (May and Wise, 1984; Kline et al., 1985; Wise et al., 1988). To quantify several

PAHs that have low fluorescence sensitivity and/or selectivity, six additional samples of SRM

1974a were extracted and prepared as described above. The extract was then fractionated on a

semi-preparative aminopropylsilane column to isolate isomeric PAH fractions as described

previously (May and Wise, 1984; Kline et al., 1985; Wise et al., 1988). These isomeric PAH

fractions were analyzed by reversed-phase LC-Fl on a similar C18 column.

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For both the GC/MS and LC-Fl measurements, selected perdeuterated PAHs were added to the

mussel tissue/sodium sulfate mixture immediately prior to Soxhlet extraction for use as

internal standards for quantification purposes. For GC/MS analyses the following internal

standards were used: naphthalene-d8, acenaphthene-d10, fluorene-d10, phenanthrene-d10,

fluoranthene-d10, benz[a]anthracene-d12, benzo[e]pyrene-d12, dibenz[a,h]anthracene-d14, and

benzo[ghi]perylene-d12. For the LC-Fl analyses the following internal standards were used:

naphthalene-d8, phenanthrene-d10, fluoranthene-d10, and perylene-d12 for the LC-Fl (Total)

analyses and triphenylene-d12, benz[a]anthracene-d12, and benzo[ghi]perylene-d14 for the LCFl (Fraction) analyses. Calibration response factors for the analytes relative to the internal

standards were determined by analyzing aliquot of gravimetrically prepared mixtures of SRMs

1491 (Aromatic Hydrocarbons in Hexane/Toluene) and 2260 (Aromatic Hydrocarbons in

Toluene, Nominal Concentration 60 μg/mL) in the case of GC/MS analyses or SRM 1647b

(Priority Pollutant PAHs in Acetonitrile) in the case of LC-Fl analyses; gravimetrically

prepared solutions of additional analytes not contained in SRMs 1491, 2260, or 1647b; and the

internal standards.

3.5. Polychlorinated biphenyl congeners and chlorinated pesticides

SRM 1974a was analyzed for selected PCB congeners and chlorinated pesticides using GC-ECD

on two columns with different selectivity and using GC/MS. This same approach has been used

previously at NIST for the certification of PCB congeners and chlorinated pesticides in other

environmental matrices, including SRM 1941a as described above.

For SRM 1974a, two sets of GC-ECD analyses, designated as GC-ECD (I) and GC-ECD (II), were

performed using similar procedures. For the GC-ECD (I) analyses, subsamples of 14 g to 16 g

(wet basis) from eight bottles were mixed with 100 g of precleaned sodium sulfate and Soxhlet

extracted for 18 hr using 250 mL of dichloromethane. Size exclusion chromatography on a

preparative-scale divinylbenzene-polystyrene column with dichloromethane as the mobile phase

was used to remove the majority of the lipid and biogenic material. The concentrated eluent

was then fractionated on a semi-preparative aminopropylsilane column to isolate two fractions

containing (1) the PCB congeners and lower polarity pesticides using hexane as the mobile phase

and (2) the more polar pesticides using 5:95 (volume ratio) dichloromethane in hexane. GC-ECD

analyses of the two fractions were performed on two columns of different selectivities for PCB

separations: 0.25 mm x 60 m fused silica capillary column with a 5% phenyl-substituted

methylpolysiloxane phase (0.25 μm film thickness) (DB-5, J&W Scientific, Folsom, CA) and a

0.32 mm x 100 m fused silica capillary column with a dimethylpolysiloxane phase containing

50% methyl C18 (0.1 μm film thickness) (CPSil 5 C18 CB, Chrompack International,

Middelburg, The Netherlands). For the GC-ECD (II) analyses, subsamples of 14 g to 16 g (wet

basis) from three bottles of SRM 1974a were extracted and one half of each extract was

analyzed using the same procedure as described above for GC-ECD (I). However, these

extractions and analyses were performed as part of three different sample sets at different

times using different calibrations, method blanks, and quality control samples for each set.

For the GC/MS analyses of SRM 1974a, subsamples of 14 g to 16 g (wet basis) each from eight

randomly selected bottles were Soxhlet extracted for 18 hr using 1:1 (volume ratio)

hexane:acetone. The extracts were concentrated and placed on a precleaned silica SPE column

and eluted with 15 mL of 1:10 (volume ratio) dichloromethane in hexane. The SPE cleanup

procedure was performed sequentially three times on separate SPE columns. The final fraction

was analyzed by GC/MS on a 5% phenyl-substituted methylpolysiloxane phase as described

above for PAH measurements.

191
For both the GC-ECD and GC-MS analyses, two PCB congeners that are not significantly present

in the mussel tissue extract (PCB 103 and PCB 198), octachloronaphthalene, 4,4'-DDT-d8, and

endosulfan-d4 were added to the mussel tissue/sodium sulfate mixture prior to extraction for

use as internal standards for quantification purposes. Calibration curves for the analytes

relative to the internal standards were prepared by analyzing gravimetrically prepared

mixtures of SRMs 2261 (Chlorinated Pesticides in Hexane, Nominal Concentration 2 μg/mL),

SRM 2262 (Chlorinated Biphenyl Congeners in 2,2,4-Trimethylpentane, Nominal Concentration

2 μg/mL); gravimetrically prepared solutions of additional analytes not contained in SRMs

2261 and 2262; and the internal standards.

In addition to the analyses performed at NIST described above, SRM 1974a was used in two

interlaboratory comparison exercises as part of the NIST Intercomparison Exercise Program

for Organic Contaminants in the Marine Environment. The results from these two exercises,

which were conducted in 1993 and 1994 and included results from 22 and 19 laboratories,

respectively, were also used in the determination of the certified values for selected PCB

congeners and chlorinated pesticides. The laboratories participating in these exercises used the

analytical procedures routinely used in their laboratories to measure PCB congeners and

chlorinated pesticides.

3.6. Methylmercury

The certified values for methylmercury are based on results of analyses of SRM 1974a and

SRM 2974 at NIST and two other laboratories: Institute of Applied Physical Chemistry,

Research Centre of Jülich, (Jülich, Germany) and the Marine Environment Laboratory,

International Atomic Energy Agency (Monaco). The results from the analyses of both SRMs

(i.e., the frozen tissue and the freeze-dried tissue) were combined to provide one certified

value for both materials.

For the determination of methylmercury SRM 1974a and SRM 2974 were analyzed at NIST

using liquid-solid extraction under acidic conditions and size exclusion chromatography followed

by GC with atomic emission detection (GC-AED) as described in detail by Donais et al. (1996;

1997). The GC-AED analyses were performed using a 0.53 mm x 15 m fused silica capillary

column with a 14% (mole fraction expressed as percent) cyanopropyl-substituted

methylpolysiloxane phase (3.0 μm film thickness) (OV-1701, Quadrex, New Haven, CT). The

method used at IAEA consisted of saponification at 70 °C followed by ethylation-room

temperature precollection-GC-pyrolysis-cold vapor atomic fluorescence spectrometric

detection (Horvat et al., 1993). At the Research Centre of Jülich the analytical procedure for

methylmercury consisted of a water steam distillation under acid conditions, anion exchange

chromatographic separation of inorganic and methylmercury followed by cold vapor atomic

absorption spectrometric detection before and after ultraviolet radiation (May et al., 1987;

Ahmed et al., 1987; Padberg et al., 1993). Subsamples from six bottles each of SRM 1974a

and SRM 2974 were analyzed by each of the three laboratories.

3.7. Certified and noncertified concentrations

Statistically combining the results from the different methods, SRM 1974a has been issued

with certified values for 15 PAHs (Table 1), 20 PCB congeners (Table 2), and 7 chlorinated

pesticides (Table 3). Noncertified values are available for an additional 18 PAHs (Table 4), 4

PCB congeners (Table 5), and 4 chlorinated pesticides (Table 5). In addition to the organic

contaminants, the methylmercury content was determined to be 77.2 ± 3.8 μg/kg dry mass

basis (Donais et al., 1997). Also, SRM 1974a was analyzed for 28 inorganic constituents using

instrumental neutron activation analysis (Schantz et al., 1997).

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4. CONCLUSION

The materials replacing SRM 1941 and SRM 1974 (SRM 1941a and SRM 1974a) do have an

increased number of compounds certified with the uncertainties generally lower for those

compounds that were certified in the original materials. An increased amount of sediment and

an increased number of mussels were collected to expand the supply for an additional period of

time. SRM 1941a is a dry sediment and SRM 1974a is a frozen mussel tissue, both of which

have concentrations of the organic contaminants typical of an urban harbor environment.

5. ACKNOWLEDGMENTS AND DISCLAIMER

The collection, preparation, and certification of SRM 1941a were supported in part by the

Coastal Monitoring and Bioeffects Assessment Division, National Ocean Service, National

Oceanic and Atmospheric Administration. The sediment material used for SRM 1941a was

collected with the assistance of the U.S. Coast Guard. M. P. Cronise and C. N. Fales from the

NIST Standard Reference Materials Program assisted in the collection of the sediment and

prepared the material. R. E. Rebbert performed the homogeneity assessment measurements. T.

L. Wade (Geochemical and Environmental Research Group, Texas A&M University) provided

some of the measurements for percent total organic carbon.

The mussel tissue material used for SRM 1974a was collected with the assistance of Julie

Seavey from Battelle Ocean Sciences Laboratory, Duxbury, MA and M. Cronise and C. Fales

from the NIST Standard Reference Materials Program. The support aspects for the preparation

and certification of this SRM were coordinated by J. C. Colbert of the NIST Standard Reference

Materials Program.

Certain commercial equipment, instruments, or materials are identified in this report to

specify adequately the experimental procedure. Such identification does not imply

recommendation or endorsement by the National Institute of Standards and Technology, nor

does it imply that the materials or equipment identified are the best available for the purpose.

6. REFERENCES

Ahmed, R., K. May, and M. Stoeppler (1987) Ultratrace analysis of mercury and

methylmercury (MM) in rain water using cold vapour atomic absorption spectrometry.

Fresenius J. Anal. Chem., 326: 510-6.

Ballschmiter, K. and M. Zell (1980) Analysis of polychlorinated biphenyls (PCB) by glass

capillary gas chromatography. Fresenius Z. Anal. Chem., 302:20-31.

Donais (Behlke), M. K., P. C. Uden, M. M. Schantz, and S. A. Wise (1996) Development,

validation, and application of a method for quantification of methylmercury in biological marine

materials using gas chromatography atomic emission detection. Anal. Chem., 68:3859-66.

Donais, M. K., R. Saraswati, E. A. Mackay, R. Demiralp, B. J. Porter, M. Vangel, M. Levenson,

V. Mandic, S. Azemard, M. Horvat, K. May, H. Emons, and S. A. Wise (1997) Certification of

three mussel tissue standard reference materials for methylmercury and total mercury

content. Fresenius J. Anal. Chem., in press.

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Horvat, M., L. Liang, and N. S. Bloom (1993) Comparison of distillation with other current

isolation methods for the determination of methylmercury compounds in low level

environmental samples. Anal. Chim. Acta, 282:153-68.

International Standards Organization (1993) Guide to the Expression of Uncertainty in

Measurement. First edition. International Standards Organization (ISO), Geneva, Switzerland.

101 pp. See also: Taylor, B. N. and C. E. Kuyatt (1994) NIST Technical Note 1297. 20 pp.

Kline, W. F., S. A. Wise, and W. E. May (1985) The application of perdeuterated polycyclic

aromatic hydrocarbons (PAH) as internal standards for the liquid chromatographic

determination of PAH in petroleum crude oil and other complex mixtures. J. Liquid

Chromatogr., 8:223-37.

May, K., M. Stoeppler, and K. Reisinger (1987) Studies of the ratio of total mercury /

methylmercury in the aquatic food chain. Toxicol. Environ. Chem., 13:153-159.

May, W. E., and S. A. Wise (1984) Liquid chromatographic determination of polycyclic aromatic

hydrocarbons in air particulate extracts. Anal. Chem., 49:225-32.

Padberg, S., M. Burow, and M. Stoeppler (1993) Methylmercury determination in

environmental and biological reference and other materials by quality control with certified

reference materials. Fresenius J. Anal. Chem., 346:686-8.

Schantz, M. M., B. A. Benner, Jr., M. J. Hays, W. R. Kelly, R. D. Vocke, Jr., R. Demiralp, R. R.

Greenberg, S. B. Schiller, G. G. Lauenstein, and S. A. Wise (1995) Certification of Standard

Reference Material (SRM) 1941a, Organics in Marine Sediment. Fresenius J. Anal. Chem., 352:

166-73.

Schantz, M. M., B. A. Benner, Jr., S. N. Chesler, B. J. Koster, K. E. Hehn, S. F. Stone, W. R.

Kelly, R. Zeisler, and S. A. Wise (1990) Preparation and analysis of marine sediment reference

material for the determination of trace organic constituents. Fresenius J. Anal. Chem., 338:

501-14.

Schantz, M. M., R. Demiralp, R. R. Greenberg, M. J. Hays, R. M. Parris, B. J. Porter, D. L.

Poster, L. C. Sander, K. S. Sharpless, and S. A. Wise (1997) Certification of a frozen mussel

tissue Standard Reference Material (SRM 1974a) for trace organic constituents. Fresenius J.

Anal. Chem., in press.

Schantz, M. M., R. M. Parris, J. Kurz, K. Ballschmiter, and S. A. Wise (1993) Comparison of

methods for the gas-chromatographic determination of PCB congeners and chlorinated

pesticides in marine reference materials. Fresenius J. Anal. Chem., 346:766-78.

Schantz, M.M., B.J. Koster, L.M. Oakley, and S.B. Schiller (1995) Certification of

polychlorinated biphenyl congeners and chlorinated pesticides in a whale blubber Standard

Reference Material. Anal. Chem., 34:901-910.

Schulte, E. and R. Malisch (1983) Berechnung der wahren PCB-gehalte in umweltproben.

Fresenius Z. Anal. Chem., 314:545-51.

Wise, S. A., B. A. Benner, Jr., R. G. Christenson, B. J. Koster, J. Kurz, M. M. Schantz, and R.

Zeisler (1991) Preparation and analysis of a frozen mussel tissue reference material for the

determination of trace organic constituents. Environ. Sci. Technol., 25:1695-704.

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Wise, S. A., M. M. Schantz, B. A. Benner, Jr., M. J. Hays, and S. B. Schiller (1995)

Certification of polycyclic aromatic hydrocarbons in a marine sediment standard reference

material. Anal. Chem., 67:1171-78.

Wise, S. A., S. N. Chesler, H. S. Hertz, L. R. Hilpert, and W. E. May (1977) Chemically-bonded

aminosilane stationary phase for the high performance liquid chromatographic separation of

polynuclear aromatic hydrocarbons. Anal. Chem., 49:2306-10.

Wise, S. A., B. A. Benner, Jr., G. D. Byrd, S. N. Chesler, R. E. Rebbert, and M. M. Schantz

(1988) Determination of polycyclic aromatic hydrocarbons in a coal tar standard reference

material. Anal. Chem., 60:887-94.

Zeisler, R., J. K. Langland, and S. H. Harrison (1983) Cryogenic homogenization of biological

tissues. Anal. Chem., 55:2431-34.

195
196
HISTOPATHOLOGY

197
Histopathology Analysis

M. S. Ellis, R. D. Barber, R. E. Hillman,* Y. Kim and E. N. Powell

Haskin Shellfish Research Laboratory

Rutgers University

6959 Miller Ave.

Port Norris, NJ

ABSTRACT

This document describes the procedures followed for histopathological analysis of

oysters, mussels, and zebra mussels collected for NOAA's National Status and Trends

Mussel Watch Program. Analyses are conducted on paraffin-embedded tissues

sectioned at a 5-μm thickness and stained using a pentachrome staining procedure.

Infection intensity of parasites, the occurrence and extensiveness of tissue

pathologies, and the intensity of diseases are recorded using semi-quantitative or

quantitative measures.

1. INTRODUCTION

The use of bivalves in the National Status and Trends (NS&T) Program is predicated upon their

reliability as environmental integrators of contamination. The influence of population health on

body burden and, in turn, the influence of contaminant exposure on population health is, as yet,

poorly understood. Clearly, certain diseases (normally caused by viruses or single-celled

prokaryotes and eukaryotes) and non-disease causing parasites (mostly ciliates and multicellular parasites) produce tissue level changes that might be expected to affect contaminant

body burden. Gonadal quantity, for example, can be dramatically altered by disease (Hofmann

et al., 1995; Barber, 1996; Ford and Figueras, 1988) and by parasites (Hopkins, 1957; Yoo

and Kajihara, 1985). Certain contaminants are preferentially concentrated in gonadal tissue

(Ellis et al., 1993; Lee, 1993; Abbe et al., 1994). Others are concentrated in non-gonadal

tissue (Mo and Neilson, 1993; Cunningham and Tripp, 1975). Because gametic material can

account for 20 to 50% of body weight in target species of oysters and mussels (Sprung, 1991;

Choi et al., 1993), the relative proportion of gonadal to somatic tissue and the timing of

spawns (an important depuration route for some contaminants) can significantly impact the

body burden of contaminants. Evidence, mostly by correlation, continues to mount for a

relationship between certain tissue pathologies and contaminant exposure (Bowmer et al.,

1991; Weis et al., 1995; Johnson et al., 1992) and the influence of contaminant exposure on

the bivalve immune system has been described (Anderson et al., 1992; Cheng, 1988; Winstead

and Couch, 1988). Besides contaminants, other environmental factors may facilitate disease or

trigger the development of pathologies (e.g. Lee et al., 1996; Landsberg, 1996) with significant

consequences to tissue composition and, in all likelihood, subsequent contaminant retention.

Thus, contaminant exposure and health, as they feed back upon each other, may have dramatic

effects on monitoring programs that use sentinel organisms to define spatial and temporal

trends in contamination loadings and contaminant gradients.

Evaluation of the health of specimens collected as a part of the Mussel Watch Program

necessitates evaluating the prevalence and intensity of diseases, parasites and pathologies by

histological examination. Certain pathological conditions recognized by shell condition

(Warburton, 1958; Lawler and Aldrich, 1987), ligament degradation (Dungan et al., 1989), or

*

 Battelle Ocean Sciences, Environmental Assessment Division, Duxbury, MA 02332.

198
periostracal abnormalities (Davis and Barber, 1994) will not be identified using this approach

and some ectoparasites are lost during collection (e.g. odostomians, White et al., 1985, 1989),

however most common diseases, parasites, and pathologies can be evaluated by this method.

Classically, histological examination involves evaluating samples for parasite prevalence and

the occurrence of pathologies. Measures of prevalence or occurrence, however, do not give a

true indication of the health of an organism. Kennicutt (1995) compared the usefulness of

prevalence to semi-quantitative and quantitative measures of intensity in the Gulf of Mexico

Offshore Operations Monitoring Experiment and found that most significant trends were

observed from intensity data rather than prevalence. One reason for this is that prevalence

depends on transmission, and transmission rate may be controlled by biological factors such as

population density, encounter rates, and inherent differences in susceptibility, as well as

factors acting as stressors directly on the individuals in the population (Kermack and

McKendrick, 1991; Ackerman et al., 1984; Hofmann et al., 1995; Powell et al., 1996). Thus,

measures of intensity or extensiveness of tissue alteration may more reliably correspond to

measures of exposure. Accordingly, beginning in 1995, an histopathology analysis was included

in the Mussel Watch Program designed to evaluate population health and the approach taken was

to evaluate the intensity of diseases and parasites, and the extensiveness of tissue pathologies,

rather than simply prevalence, to better assess the health of sampled populations.

A measure of overall health has not been applied. A number of measures of overall health have

been suggested, based on tissue appearance (Quick and Mackin, 1971), histological grading

(Bowmer et al., 1991), or summation of total parasite load (Kennicutt, 1995). Although Laird

(1961) argues on theoretical grounds for an underlying relationship between total parasite

body burden and environmental quality, generally, these overall measures of health have not

proven efficacious because the various parasites, diseases and pathologies originate in

different ways and certain parasites, even if abundant, may not have a large impact on

organism health.

2. EQUIPMENT, REAGENTS, SOLUTIONS, AND SAMPLE PREPARATION

Preparation of samples for histopathological analysis follows the protocols established for

gonadal analysis (see Ellis et al., this volume).

3. ANALYSIS

Prepared slides are examined individually under the microscope using a 10X ocular and a 10X

objective. If any tissue needs to be examined more closely, a 25X or 40X objective may be

used for closer examination of suspected pathologies or parasites. Major tissue types to be

examined include gill, mantle, gonad and gonoducts, digestive gland tubules, stomach/digestive

gland ducts, and connective tissue. Thus, a proper tissue cross-section is essential (see Ellis et

al., this volume). Normally, oysters are examined starting at the gills, and scanned laterally,

working toward the anterior end. In mussels, gonads and gills are examined first, and then the

visceral mass. For zebra mussels, the visceral mass, including the gonads and digestive tract

are examined, followed by scanning of the gills and mantle.

Conditions evaluated, be they parasites, diseases, or tissue pathologies, are scored for

intensity using either a quantitative or semi-quantitative scale, as described subsequently.

Conditions scored quantitatively are evaluated by keeping a running count of incidences of the

condition as the slide is scanned, to avoid re-examining each slide multiple times for each

separate malady. Evaluation of conditions scored semi-quantitatively may require re-scanning

199
portions of the tissue for each malady type to gain a complete portrayal of the degree of tissue

involvement.

We list below the common conditions encountered during the first few years of

histopathological analysis, including, in each case, the method of analysis, a short description,

and a reference to published figures of the condition. The list is not intended to be inclusive of

all known conditions for these species or of all conditions encountered in specimens obtained

during the Mussel Watch Program. Frequently, in routine examination, we have not attempted

to differentiate, at a low taxonomic level, between related parasites because infection

intensities are low for the majority of parasite species and, so, the information gained from

taxonomic analysis does not warrant the time spent in identification. Rather, we have lumped

the various species into higher categories (e.g. all cestodes, all ciliates). When further

differentiation is needed, we have first differentiated by tissue of occurrence because most

species have distinct tissue preferences. In nearly all cases, this level of differentiation has

been adequate for estimates of prevalence and infection intensity.

3.1. Quantitative categories

Most parasites were counted quantitatively. These include Nematopsis spp., ciliates of various

types, worms in the gonoducts, worms ectoparasitic or commensal on the gills, cestodes,

nematodes, and copepods. We also evaluated a number of other tissue conditions quantitatively:

the number of ceroid bodies, incidences of tissue inflammation, rickettsial bodies, incidences of

tissue edema, and suspected neoplasms and tumors.

Nematopsis [Photograph: Cheng (1967), p. 148, Fig. 29; Ford (1988), p. 218, Fig. 6G; Gauthier

et al. (1990), p. 110, Fig. 2, 3] is a sporozoan parasite normally found in oysters, and only

rarely in mussels and zebra mussels. Different Nematopsis species often have different tissue

distributions, and, in particular, show a tissue preferences for either mantle or gill (Sprague

and Orr, 1952). Although, species identifications were not conducted, we routinely scored gill

and mantle infection intensity separately, following Landau and Galtsoff (1951). Quantification

was obtained by counting each individual in each tissue within one representative tissue

section.

Ceroid bodies or brown cells [Photograph: Murchelano and MacLean (1990), p. 10, Fig. 1- 9],

distinct brown-yellow aggregates that may occur in large clumps, appear to be involved in

metabolite accumulation and detoxification (Zaroogian and Yevich, 1993). Typically, they occur

in greatest abundance in oysters and in lesser numbers in mussels and zebra mussels.

Quantification was obtained by counting each ceroid body. Occasionally, a ceroid body appears

fractured or split; in this case only one fragment is counted.

A variety of kinds of ciliates have been observed, normally at low prevalence and more

frequently in mytilid mussels than in oysters. Ciliates have been quantitated by tissue type

(e.g. gut, digestive gland, gill) and by size [e.g. individual ciliates versus xenomas (cells

distended with maturing ciliates) or larger attached ciliates]. A more detailed taxonomic

differentiation can be conducted if high infection intensity warrants it, but this is rarely the

case. If the large xenoma has not burst, it is counted as an individual large ciliate; if it has

burst, the individual small ciliates are counted. [Photograph: Cheng (1967), p. 184, Fig. 88,

representative gill ciliate; Cheng (1967), p. 193, Fig. 102, representative digestive gland

ciliate in lumen; Figueras et al. (1991), p. 91, Fig. 2, representative digestive gland ciliate in

tubule wall; additional photographs in Gauthier et al. (1990)]

200
Figure 1. Unidentified worm in the gonoduct of an oyster, Crassostrea virginica.

An unidentified, possibly larval arabellid, worm is occasionally seen in the gonoducts of

oysters from the Gulf of Mexico (Figure 1). The cross-section is characterized by a distinct

cuticle and, occasionally, setae appear to be present. Each occurrence is counted.

Gill nemerteans are occasionally seen between gill filaments. Whether these are commensal or

ectoparasitic is unknown. Each individual cross section is counted separately, although, like

nematodes, a single individual may be responsible for a number of cross-sections.

A variety of encysted metacestodes and trematodes have been observed, normally at low

prevalence (for examples, see Cake, 1977; Cake and Menzel, 1980). Cestodes are more

common in oysters, trematodes, except for Bucephalus, in mytilid mussels. Cellular reaction in

the subepithelial tissues to cestode cysts (e.g., encapsulation of the larval cestodes) has been

described by Sindermann (1970) and is normally observed associated with them. Cestode cysts

and trematodes have been quantitated by tissue location (e.g. gut, gill, gonad). Each occurrence

observed is counted separately. Representative examples are given in Murchelano and MacLean

(1990, p. 16, Fig. 1 - 21), Sindermann (1970, p. 128, Fig. 42), and Little et al. (1969, p. 455,

Fig. 1).

Nematodes [Photograph: Gauthier et al. (1990), p. 112, Fig. 9; Murchelano and MacLean

(1990), p. 18, Fig. 1 - 25; Sparks (1985), p. 375 - 376, Fig. 10 - 16] are occasionally found

in most tissues of oysters. Each cross-section observed is counted; like nemerteans and many

other worms, one individual may be responsible for a number of tissue cross-sections,

however this method of quantification has proven effective even at high infection intensities

(Kennicutt, 1995).

201
Figure 2. Edema in the gill of an oyster, Crassostrea virginica.

Cases of tissue inflammation [Photograph: Murchelano and MacLean (1990), p. 10, Fig. 1 - 10;

Sindermann (1970), p. 110, Fig. 34], characterized by intense infiltration of hemocytes, may

be focal or diffuse. The type of affected tissue and type of irritation responsible influence the

nature of the cellular response (Ford and Tripp, 1996). Each affected area is counted. Diffuse

inflammation is differentiated from focal inflammation when the affected area does not appear

to have a clear center or focal point of highest hemocyte concentration and hemocytes are

abundant and distributed broadly over a large section of tissue.

Amorphous, basophilic, granular inclusions, variously referred to as rickettsial bodies,

chlamydial bodies or mycoplasms, are normally observed in the duct and tubule walls of the

digestive gland [Photograph: Murchelano and MacLean (1990), p. 8, Fig. 1 - 6; Harshbarger et

al. (1977), p. 667, Fig. 1]. Each individual inclusion is tabulated.

Neoplasms and tumors are occasionally observed, particularly in mytilid mussels where

disseminated sarcomas, probably of hematopoietic origin, are particularly common in the Puget

Sound region (e.g. Elston et al., 1990). Occurrence of neoplasms and tumors in oysters is

extremely rare. Examples are described by Farley (1969, 1976), Harshbarger et al. (1979)

and Ford and Tripp (1996). Representative photographs are provided by Murchelano and

MacLean (1990, p. 19 - 20, Fig. 1 - 27, 1 - 29), Peters (1988, p. 81, Fig. C, E, F), and Sparks

(1985, p. 107, Fig. 27; p. 109, p. 120, Fig. 51; p. 113, Fig. 42). Each occurrence is tabulated.

Edema (Figures 2, 3) is a localized enlargement of tissue cells without an accompanying

response by hemocytes or if hemocytes are present, they too appear to be enlarged. Each

affected area is counted separately.

202
Figure 3. Edema in the connective tissue of an oyster, Crassostrea virginica.

Proctoeces sp. [Wolf et al. (1987), p. 380, Fig. 1; see also Winstead and Couch (1981)] is

occasionally found in the gonoducts. This trematode and occasional nematodes also found in the

gonoduct should be distinguished from the more common unidentified, possibly arabellid, worm

observed in oysters from the Gulf of Mexico. Each occurrence is tabulated.

Parasitic copepods are occasionally found in the gut lumen (Gee and Davey, 1986). Each

occurrence is tabulated.

3.2. Semi-quantitative categories

Some conditions were assigned to a semi-quantitative scale related to the intensity of the

effect or the extensiveness of the affected area. With one exception, these were pathologies

affecting large tissue areas or diseases characterized by systemic affects and individual

counts too large to quantify. One invasive parasite, Bucephalus, was also included due to the

extensiveness of infections and the difficulty in obtaining quantitative counts of the sporocysts.

Perkinsus marinus was assayed in oysters by the more precise thioglycollate method, rather

than by histology (Powell and Ellis, this volume).

Digestive gland atrophy [Photograph: Gauthier et al. (1990), p. 112, Fig. 10; Winstead (1995),

p. 107, Fig. 3,4], a condition characterized by the thinning of the lumen of the digestive tubule

walls, has been described in a number of bivalve species (Bielefeld, 1991; Marigómez et al.,

1990; Axiak et al., 1988). Causes of the condition have been ascribed to a variety of stressors

including exposure to contaminants and variations in food supply. Winstead (1995) found that

poor nutrition was a key element in producing the condition in oysters and that the digestive

gland recovered to its normal state relatively rapidly once food supply improved. Presumably,

the thinning of the digestive tubule epithelium affects the normal digestive processes.

203
The digestive gland is scanned for tubules showing evidence of epithelial thinning. A visual

evaluation is made of the average condition of the digestive gland tubules and this average

condition is assigned to a semi-quantitative numerical scale (Table 1) according to the degree

of thinning of the epithelial wall. The semi-quantitative assessment permits the reading of

many samples in a short time. For increased accuracy, the ratio of tubule diameter to wall

diameter (e.g. Winstead, 1995) or a direct measure of wall thickness (Marigómez et al., 1990)

can be used.

Bucephalus sporocysts [Photograph: Gauthier et al. (1990), p. 112, Fig. 8; Murchelano and

MacLean (1990), p. 16, Fig. 1-23; Sindermann (1970), p. 125, Fig. 41] occur principally in the

gonadal tissue of oysters (Hopkins, 1957). Extensive infections effectively destroy all gametic

tissue and the sporocysts begin to invade the remainder of the visceral mass. On rare

occasions, the gill tissue may also be involved. The large branching sporocysts are difficult to

quantitatively enumerate. Hence, infection intensity is scored on a semi-quantitative scale

(Table 2).

Table 1. Semi-quantitative scale for digestive gland atrophy (adapted from scale developed by

J. Gauthier).

Score Description

0 Normal wall thickness in most tubules (0% atrophy), lumen nearly occluded, few

tubules even slightly atrophied (Figure 4)

1 Wall thickness averaging less than one-half atrophied, most tubules showing some

atrophy, some tubules still normal

2 Wall thickness averaging about one-half as thick as normal (Figure 5)

3 Wall thickness greater than one-half atrophied, most tubules walls significantly

atrophied, some walls extremely thin (fully atrophied)

4 Wall extremely thin (100% atrophied), nearly all tubules affected (Figure 6)

204
Figure 4. Crassostrea virginica normal digestive tubule, scored a 0 according to Table 1.

Figure 5. Digestive gland atrophy in Crassostrea virginica scored a 2 according to Table 1.

205
Figure 6. Digestive gland atrophy in Crassostrea virginica scored a 4 according to Table 1.

Table 2. Semi-quantitative scale for Bucephalus infection.

Score Description

0 Uninfected

1 Present in the gonads only (some gametic tissue still present) (Figure 7)

2 Completely filling the gonads (no gametic tissue present); may be present in digestive

gland or gills in very limited amounts (Figure 8)

3 Completely filling the gonads; extensive invasion of the digestive gland and/or the gills

(Figure 9)

4 Completely filling the gonad; substantially filling the digestive gland or gill; individuals

appear to be a sac of sporocysts

206
Figure 7. Bucephalus infection in Mytilus edulis scored a 1 according to Table 2. Some gametic

tissue is still present.

Figure 8. Bucephalus infection in Mytilus edulis scored a 2 according to Table 2. No gametic

tissue is present.

207
Figure 9. Bucephalus infection in Mytilus edulis scored a 3 according to Table 2. No gametic

tissue is present. Bucephalus heavily infiltrating digestive gland.

Haplosporidium nelsoni [photograph: Ford and Tripp (1996), p. 614, Fig. 18] is a haplosporidian

protozoan responsible for MSX disease in oysters. The intensity of MSX infection is graded

according to a semi-quantitative scale (Table 3) reminiscent of the scale established for

Perkinsus marinus. Because of the small size of H. nelsoni, oyster tissues may need to be

examined at a higher power than 10X, in some cases. Because MSX infection normally starts in

the gill epithelium, the gill tissue must be carefully examined to score early infections

accurately. In cases where the disease has become systemic, examination of the visceral mass

is necessary to score infection intensity. More details are provided in Ford (1985, 1986) and

Ford and Figueras (1988).

Several kinds of abnormal gonadal development have been observed, sometimes commonly, in

mytilid mussels. Often, abnormal gonadal development is characterized by unusual development

at the base of the follicles. The cells resemble that of a germinoma and are differentiated from

that of normal development by being either enlarged, or appearing to have an enlarged nucleus.

This type of abnormal development is more common in males than in females. In other cases,

underdeveloped gonadal follicles were observed. Follicles were small and occupied a smaller

portion of the mantle tissue. Ova were smaller and male follicles often had very few mature

sperm. Sometimes follicles may be filled with anomalous ova or degenerating sperm, and

granulocytes, brown cells and cellular debris may clog the follicular lumens (Figure 10).

Sometimes cells adhere to each other, forming accumulations and empty spaces among

developing cells. Occasionally, fibrosis occurs, with proliferation of fibroblasts inside the

follicles and in the interfollicular connective tissue. Abnormal gonadal development is often

associated with degeneration of Leydig tissue around the follicles and hemocytic infiltration

into the surrounding tissues (Figure 11).

208
Table 3. Semi-quantitative scale for Haplosporidium nelsoni infection [from Ford (1985, 1986)

and Ford and Figueras (1988)].

Score Description

0 Uninfected

1 Epithelial only, rare to lightly infected, 1 - 100 parasites per field of gill tissue

2 Subepithelial, spreading into the visceral mass, lightly to moderately infected with

clear subepithelial foci, normally 10 - >100 parasites per field but <5 parasites per

1000X oil field

3 Subepithelial, more evenly distributed in visceral mass, lightly to moderately infected

normally 10 - >100 parasites per field but <5 parasites per 1000X oil field

4 Heavy systemic infection, averaging more than 5 parasites per oil field

Figure 10. Mytilus edulis follicle with abnormal gametic tissue (lower right).

209
Figure 11. Mytilus edulis follicle heavily infiltrated with hemocytes (right).

The approach used to score instances of abnormal gonadal development differs from that used

for digestive gland atrophy in that the scale rates the spatial coverage of the condition (e.g.

fraction of follicles affected), but not the degree of effect in each follicle (Table 4). Normally,

the entire follicle is completely affected or not affected.

4. CONCLUSION

The described technique provides a quantitative or semi-quantitative method to determine the

prevalence and intensity of parasites, pathologies, and diseases afflicting oysters, blue

mussels and zebra mussels. The described method is a general approach for histopathological

analysis targeting a wide range of parasites and pathologies. Certain conditions will be better

assessed by other methods; Perkinsus marinus infection in oysters being a good example

(Powell and Ellis, this volume). The described method emphasizes the quantification of

intensity. Prevalence rarely provides an adequate description of the population dynamics of

disease and, in practice, often yields ambiguous results. Infection intensity quantitated by

counts or the use of a semi-quantitative scale consistently provides a more robust data set for

statistical analysis comparing the spatial and temporal distribution of parasites, pathologies,

and diseases to contaminant body burden.

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Table 4. Semi-quantitative scale for abnormal gonadal development in mussels.

Score Description

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3 More than half the follicles are affected

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reproductive capacity of the blue mussel (Mytilus edulis galloprovincialis). Bull. Korean Fish.

Soc., 18:581-5.

Zaroogian, G., and P. Yevich (1993) Effect of selected inhibitors on cadmium, nickel, and

benzo[a]pyrene uptake into brown cells of Mercenaria mercenaria. Mar. Environ. Res.,

35:41-5.

215
Gonadal Analysis

M. S. Ellis, R. D. Barber, R. E. Hillman,* and E. N. Powell

Haskin Shellfish Research Laboratory

Rutgers University

6959 Miller Ave.

Port Norris, NJ

ABSTRACT

This document describes the procedures for determining the reproductive stage of

oysters, mussels, and zebra mussels collected for NOAA's National Status and Trends

Mussel Watch Program. Procedures are documented for preparation of

paraffin-embedded tissues sectioned at a 5-μm thickness and stained using a

pentachrome staining procedure.

1. INTRODUCTION

Assessment of the physiological state of bivalve populations requires an analysis of the state

of gonadal development. Determination of reproductive stage was included as part of the Mussel

Watch Project to give an indication of the amount of gametic material in the bivalves' tissues at

the time of chemical analysis. Certain contaminants are preferentially concentrated in gonadal

tissue (Ellis et al., 1993; Lee, 1993; Abbe et al., 1994). Others are concentrated in

non-gonadal tissue (Mo and Neilson, 1993; Cunningham and Tripp 1975). Because gametic

material can account for 20% to 50% of body weight in target species of oysters and mussels

(Sprung, 1992; Choi et al., 1993), the relative proportion of gonadal to somatic tissue and the

timing of spawns (an important depuration route for some contaminants) can significantly

impact the body burden of contaminants.

The original intent of the determinations of reproductive stage was to assure that sampling was

conducted at the same stage of the reproductive cycle so that analysis of neither the lipophilic

organic contaminants nor the trace element contaminants were influenced by reproductive

state. Unfortunately, the time required for sampling and the wide latitudinal range encompassed

by the sites did not permit consistent recovery of individuals in a similar stage of reproductive

development at all sites. For example, typically, oysters are undifferentiated in the winter.

Gonads begin to develop in early spring and spawning occurs during late spring through early

fall. Most Gulf Coast oysters spawn at least twice during this time period. Single spawns tend

to occur in the shorter summers of the mid-Atlantic region. The timing of the last spawn varies

with latitude and with yearly variations in climate (e.g. Wilson et al., 1990, 1992). Certain

sites, particularly southeast Atlantic and southern Gulf sites, routinely yield oysters in

reproductive development or ready to spawn in mid-winter during the period when Mussel

Watch sampling occurs at these sites. Mid-Atlantic sites typically were characterized by

individuals in an undifferentiated state, that thus contained significantly less lipid-rich gametic

tissue and that had been accumulating contaminants for a much longer time since their most

recent spawn. Mytilids and zebra mussels had the same assortment of problems relating to

latitude and interannual changes in climate (Newell, 1989; Seed and Suchanek, 1992;

Borcherding, 1991). Thus, analysis of reproductive stage proved important in identifying

differences in tissue composition which might affect between site and interannual comparisons

*

 Battelle Ocean Sciences, Environmental Assessment Division, Duxbury, MA 02332.

216
of contaminant data. Wilson et al. (1990, 1992) discuss the influence of climate on

reproductive stage and contaminant body burden in the Mussel Watch Program in more detail.

Oyster gonadal tissue is distributed around the body mass and cannot be easily excised and

weighed (Morales-Alamo and Mann, 1989). In contrast to oysters, in mussels the gonadal

follicles develop primarily within the mantle. Zebra mussel gonads develop within the body,

around the periphery of the viscera. In none of these cases can the gonad be easily excised and

weighed. Consequently, virtually all assays of reproductive stage use histological methods to

recognize the changes in the germinal epithelium and germinal products that identify stages in

gonadal development. The histological approach uses a semiquantitative numerical assignment to

rank reproductive stage. Quantitative measures, such as a measure of egg protein content (Choi

and Powell, 1993), remain expensive and time consuming, and do not permit a concomitant

histopathological analysis. Therefore, a histological examination is still the single method of

choice, when only one method can be used.

For oysters and mytilid mussels, a dorsal-ventral slice of tissue is taken and fixed in

Davidson's fixative. Zebra mussels are decalcified and embedded whole. Tissue samples are

embedded in paraffin, sectioned, and stained using a pentachrome staining protocol. Stained

sections are examined under a compound microscope, and sex and the state of gonadal

development determined. Fixation follows the method described in Preece (1972). The staining

procedure is an adaptation by one of us (Barber) of Masson's (1928) trichrome procedure.

Reproductive stage in oysters is determined using a semiquantitative scale adapted from Ford

and Figueras (1988). The scale developed by Seed (1975, 1976) for determining gonad index in

mussels was adopted for mussels and zebra mussels.

2. EQUIPMENT, REAGENTS AND SOLUTIONS

2.1. Equipment

Stainless steel molds, various sizes. Fisher Scientific, Pittsburgh, PA.

Disposable microtome blades, 12-634-11. Fisher Scientific, Pittsburgh, PA..

Cover slips - square no. 1, 22 mm x 22 mm, 12-531-A. Fisher Scientific, Pittsburgh, PA.

Disposable cassettes - HistoPrep tissue capsules, 15-182-218. Fisher Scientific, Pittsburgh,

PA.

Paraffin embedding center, 12-653-68. Fisher Scientific, Pittsburgh, PA.

Embedding rings - HistoPrep embedding rings, 12-652-1OB. Fisher Scientific, Pittsburgh, PA.

Frosted microslides, 12-552. Fisher Scientific, Pittsburgh, PA.

Microscope, compound, Zeiss, 12-070-20. Fisher Scientific, Pittsburgh, PA.

Microtome, Leica Histocut 820 Rotary microtome, 12-603. Fisher Scientific, Pittsburgh, PA.

Microtome knife sharpener, automatic, Leica 903, 12-643-40. Fisher Scientific, Pittsburgh,

PA.

Drying oven, 13-254-1. Fisher Scientific, Pittsburgh, PA.

Paraffin pitcher, electric, Lepshaw 220, M7395. Baxter Scientific, McGaw Park, IL.

Rotary tissue processor, Tissue-Tek, M7286-1. Baxter Scientific, McGaw Park, IL.

Slide warmer, 12-594. Fisher Scientific, Pittsburgh, PA.

Staining dishes, 08-813A. Fisher Scientific, Pittsburgh, PA.

Vacuum oven, 13-264A. Baxter Scientific, McGaw Park, IL.

Water bath - Fisher Tissue PrepModel 134 Flotation Bath, 15-464. Fisher Scientific,

Pittsburgh, PA.

217
2.2. Reagents

Acid fuchsin, certified stain (C20H17N3Na2O9S3) [3244-88-0], A3908. Sigma Chemical Co.,St.

Louis, MO.

Ammonium hydroxide (NH4OH) [1336-21-6], A6899. Sigma Chemical Co., St. Louis, MO.

Aniline blue powder, certified stain [28631-66-5], A697-25. Fisher Scientific, Pittsburgh,

PA.

Chromotrope powder 2R, (C16H10N2Na2O8S2) [4197-07-3], C3143. Sigma Chemical Co., St.

Louis, MO.

Ethanol (C2H6O) [64-17-5], R8382. Sigma Chemical Co., St. Louis, MO.

Fast green FCF, certified stain (C37H34N2O10S3Na2) [2353-45-9], F7252. Sigma Chemical Co.,

St. Louis, MO.

Ferric ammonium sulfate [NH4Fe(SO4)2

.

 12H2O] [7783-83-7], F1018. Sigma Chemical Co., St.

Louis, MO.

Formaldehyde, 37% solution (CH2O) [50-00-0], F1635. Sigma Chemical Co., St. Louis, MO.

Glacial acetic acid (C2H402) [64-19-7], A0808. Sigma Chemical Co., St. Louis, MO.

Glycerin (C3H803) [56-81-5], G7893. Sigma Chemical Co., St. Louis, MO.

Hematoxylin powder, certified stain (C16H1406) [517-28-2], H3136. Sigma Chemical Co., St.

Louis, MO.

Hydrochloric acid, 12 N, (HCI) [7647-01-0], H7020. Sigma Chemical Co., St. Louis, MO.

Orange G powder, certified stain (C16H10N2O7S2Na2) [1936-15-8], 07252. Sigma Chemical

Co., St. Louis, MO.

Paraffin - Paraplast tissue embedding media (melting pt. 56 °C), 12-464-11, Fisher Scientific,

Pittsburgh, PA.

Permount mounting media, SP15-500. Fisher Scientific, Pittsburgh, PA.

Phosphomolybdic acid (24MoO3

.

 P2O5

.

 H2O) [51429-74-4], P7390. Sigma Chemical Co., St.

Louis, MO.

Phosphotungstic acid (12WO3

.

 H3PO4

.

 H2O) [12501-23-4], P4006. Sigma Chemical Co., St.

Louis, MO.

Sodium chloride (NaCI) [7647-14-5], S9625. Sigma Chemical Co., St. Louis, MO.

Sulfuric acid (H2SO4) [7664-93-9], S1526. Sigma Chemical Co., St. Louis, MO.

Tissue Clear II, SH2-4. Fisher Scientific, Pittsburgh, PA.

Tissue Dry, SH5-20. Fisher Scientific, Pittsburgh, PA.

Xylenes, histological grade (C8H10), X35-4. Sigma Chemical Co., St. Louis, MO.

2.3. Solutions

Ferric alum mordant: 25 g ferric ammonium sulfate dissolved in 500 mL distilled water.

Basic ethanol: 26 mL ammonium hydroxide in a solution of 3370 mL 95% ethanol and 630 mL

distilled water.

Phosphomolybdic acid solution: 5 g phosphomolybdic acid dissolved in 495 mL distilled water.

1% acetic acid: 20 mL glacial acetic acid in 1980 mL distilled water.

1% acid acetone: 20 mL acetone in 1980 mL distilled water

Groat/Weigert hematoxylin working solution: 245 mL distilled water, 5 mL sulfuric acid, 5 g

ferric ammonium sulfate, 245 mL 95% ethanol, and 2.5 g hematoxylin powder.

218
Acid fuchsin working solution: 2.5 g acid fuchsin powder dissolved in 495 mL distilled water, to

which is added 5 mL glacial acetic acid.

Phosphotungstic acid solution: 10 g phosphotungstic acid dissolved in 490 mL distilled water.

Orange G - stock: 4 g orange G powder dissolved in 495 mL distilled water to which was added

5 mL glacial acetic acid.

Chromotrope 2R - stock: 3 g chromotrope powder dissolved in 495 mL distilled water to which

was added 5 mL glacial acetic acid.

Orange G/Chromotrope working stock: 400 mL orange G solution and 100 mL chromotrope

solution.

Fast green - stock: 8 g fast green FCF dissolved in 495 mL distilled water to which was added 5

mL glacial acetic acid.

Aniline blue - stock: 6 g aniline blue powder in 495 mL distilled water.

Fast green/Aniline blue working solution: 150 mL fast green solution and 350 mL aniline blue

solution.

Davidson's fixative stock solution: 1 part glycerin, 2 parts 37 - 40% formaldehyde, 3 parts

95% ethanol, and 3 parts isotonic sodium chloride (usually 20 - 30o/ oo). The solution was

stored at 4 °C.

Davidson's fixative working solution: Just before use, 1 part of glacial acetic acid was mixed

with 9 parts of the stock solution.

3. SAMPLE COLLECTION AND FIXATION

3.1. Sampling

From 1986 to 1994, the same oysters were used for organic contaminant analysis and gonadal

analysis at all Gulf coast sites (Powell et al., 1993). The use of the same animals for gonadal

analysis and for the analysis of contaminant body burden potentially biases the latter analyses

because digestive gland tissue and gonadal tissue, which contribute a disproportionate portion

of the tissue taken for histological analysis, may contain a higher than average body burden of

certain contaminants. Sericano et al. (1993) showed that this source of error resulted in an

underestimation of true body burden by no more than 10% under most conditions for the large

oysters used in the Mussel Watch Program, if the slice of tissue removed was limited to a 3 to

5 mm section. The potential error would be much larger for the smaller mytilids and zebra

mussels. In order to avoid this error, separate samples were obtained for the gonadal analysis

portion of the Mussel Watch project for these species (and East coast oysters) in all years,

and, beginning in 1995, the same protocol was adopted for the Gulf coast oysters. Thus, the

present sampling method requires the assumption in all cases that individuals collected from a

common collection area will have experienced similar chemical loading and therefore avoid the

error introduced by subsampling the tissue to be analyzed for contaminants.

219
0 1 2 3 4 5

cm

Gill

Mouth

Labial palps

Shell

Tissue taken for

gonadal analysis

Anus

Digestive gland

Figure 1. Oyster tissue used for quantifying reproductive stage (adapted from Galtsoff,

1964).

3.2. Tissue preparation

3.2.1. Oyster tissue preparation

Each oyster was opened with an oyster knife by cutting the adductor muscle at its connection

with the upper shell. A 3- to 5-mm thick cross-section of tissue was removed from the oyster

using a scalpel or scissors (Figure 1). The determination of reproductive stage is based on a

histological evaluation of the maturation stage of oyster gonads, which are located around the

visceral mass. The tissue section is obtained such that the dorsal-ventral aspect passes

through the digestive gland and gill tissue just posterior to the palps. This aspect provides

example sections of most oyster tissues for histopathological analysis (Ellis et al., this

volume), while also providing a representative cross-section of oyster gonad.

Each section was immediately placed in a tissue cassette and the cassette placed in a jar filled

with Davidson's fixative for 48 hr. Bouin's fixative is an adequate alternative in most

applications. After 48 hr, the fixative was decanted, 50% ethanol added and the tissues

220
allowed to stand for 2 hr, during which time the vials are occasionally shaken. After 2 hr, the

50% ethanol solution was replaced with 70% ethanol, and the vials shaken every few hours.

After 24 hr, the 70% ethanol was replaced with fresh 70% ethanol for storage until

processing.

3.2.2. Mussel tissue preparation

Determination of reproductive stage for mussels was based on a histological evaluation of the

maturation stage of mussel gonads, most of which are located in the mantle (Newell, 1989).

Ordinary means of removal of mussels from their shells usually resulted in severe damage to

the mantle tissue lying next to the shell. Therefore, it was easier to preserve the mussels in

their shells. Once preserved, the tissue hardened and become easier to dissect.

The tip of a sharp knife was carefully inserted between the shells at the ventral lip and run

dorsally between the shells until the adductor muscle cut so that the shells remain in an open

position. Care was taken to cut no further than the adductor muscle to avoid cutting into the

digestive gland immediately below the adductor muscle. The mussels were then placed in a

wide-mouth jar filled with Davidson's fixative. Because the entire animal was being preserved,

the specimens were usually left in fixative for at least a week to ensure preservation of all

tissues. After this time, the fixative was decanted, 50% ethanol added, and the tissue allowed

to stand for 48 hr, during which time the jar was shaken occasionally. The solution was then

poured off, 70% ethanol added and the mixture allowed to stand for 72 hr, after which the 70%

ethanol can be replaced with fresh 70% ethanol for storage until processing.

To excise the mussel meat from its shell, a scalpel was carefully run between the mantle and

the lip of the shell, detaching the mantle from the shell. This was done around the

circumference of one of the shells and the scalpel then used to carefully separate the meat from

the shell. This procedure was repeated for the other shell. A 3 - 5 mm thick cross-section,

obtained such that the dorsal-ventral aspect passes through the digestive gland at an angle

across the body slightly toward the forward edge, was then removed using a scalpel or

scissors. Each section was placed in a tissue cassette and processed immediately after

dissection.

If the mantle tissue was damaged during the shell-opening procedure, the specimen was

discarded because the wound could result in the loss of gametic material, and lead to an

erroneous evaluation. Some damage to the gills and viscera was acceptable, but care should be

taken to minimize that damage, so the tissues may be used for histopathological evaluation

(Ellis et al., this volume) as well as determination of reproductive stage.

3.2.3. Zebra mussel preparation

Most of the gonad of a zebra mussel was concentrated within the visceral mass (Borcherding,

1990). Due to their small size however, dissection of living tissue without destroying the

gonads is difficult. Therefore, zebra mussels were preserved whole, in their shells. The

animals were placed whole, without bothering to cut the adductor muscle, into a jar with

Davidson's fixative and left in fixative for one week to allow adequate time for tissue fixation.

After this time, 20 to 30 mL of acetic acid was added to enhance decalcification of the shell.

The shell was properly decalcified when examination by hand showed that the shell is no longer

hard.

After decalcification was complete, the Davidson's was replaced with ethanol according to the

procedure followed for mussels. Once the zebra mussels were placed in the final 70% ethanol

solution, they were ready for embedding. Prior to embedding, a small tear was made in a

section of the mantle away from the visceral mass to prevent air bubbles from forming within

221
the body cavity which limits proper infiltration of the paraffin. Byssal threads must also be cut

away from the byssal gland to prevent problems when sectioning the tissue.

4. SLIDE PREPARATION

4.1. Tissue embedding

Individual tissue samples were prepared for embedding in paraffin using an established

dehydration protocol (Table 1). The solutions used for dehydration, clearing, and infiltration

were changed frequently to maintain solution purity.

The tissue embedding sequence used an automated tissue processor which automatically

processes tissue through the dehydration-clearing series and into paraffin. Embedding can also

be done manually by moving the tissues through the sequence. The paraffin was melted in an

oven set at no more than 2 °C above the melting temperature and kept at temperatures no

higher than 2 °C above melting during the infiltration process. Newly-melted paraffin should

always be used in the final infiltration and embedding steps.

After the tissues were infiltrated with paraffin (Table 1), they were transferred to a paraffin

pot and placed under a vacuum for 30 min. The vacuum oven was set to maintain the paraffin at

the same carefully-controlled temperature. The tissues were then transferred to stainless

steel molds filled with newly-melted paraffin. Oysters and mussels were oriented

cross-sectional face down for sectioning, and a plastic mold embedding ring placed on top.

Unlike the mytilids and the oysters, zebra mussels were embedded whole. Zebra mussels were

placed on their side and, when the paraffin was added, care was taken to ensure that no air

bubbles formed within the body cavity. The plastic backing was filled with paraffin and the mold

moved to a cold plate. As the tissue/paraffin cooled and hardened, the paraffin shrinks. Care

must be taken to use sufficient paraffin to cover the tissue after it cools and hardens.

Occasionally, more paraffin must be added. The mold was left on the cold plate until the

tissue-paraffin block was removed. The tissue-paraffin block was then placed in a freezer over

night before sectioning. When sectioning a number of blocks, the remaining blocks were placed

in a tray of ice to keep them cold until sectioned.

Table 1. Tissue embedding sequence*.

Dehydration Clearing

Tissue Dry 60 min Tissue Clear 60 min

Tissue Dry 120 min Tissue Clear 120 min

Tissue Dry 120 min Tissue Clear 120 min

Tissue Dry 120 min

Tissue Dry 120 min Infiltration

Tissue Dry 120 min paraffin 120 min

Tissue Dry 60 min paraffin 120 min

paraffin in vacuum oven 30 min

* In cases where the sequential solutions are the same, each transfer is a transfer to a fresh solution.

222
4.2. Tissue sectioning

The paraffin blocks were first cut at 20 μm to expose an entire tissue cross-section, and then

sliced at 5 μm using an AO microtome. Tissue sections may be cut singly, or into contiguous

sections. The tissue sections were placed on the surface of a water bath maintained at 45-50

°C and allowed to expand. Once the sections expanded to full size, a microscope slide was held

at a 45° angle and slid under one or more of the tissue sections and the sections lifted out of the

water onto the slide. The sections were positioned on the slide in the orientation in which they

will be stained and read. The slide was then placed on a slide warmer until dry or allowed to air

dry until it could be placed in a slide rack. The slide rack was placed in a drying oven overnight

at 40 °C. After drying overnight, the slides were ready to stain.

4.3. Tissue staining

The staining procedure was Barber's adaptation of the trichrome stain of Masson (1928) as

modified by Gurr (1956). Barber's modifications include the addition of aniline blue to the fast

green working solution, substitution of chromotrope 2A/orange G for Ponceau de Xylidene, and

the addition of phosphotungstic acid prior to the orange G/chromotrope stain; the procedure is

now a pentachrome technique. The addition of these stains yields better differentiation of tissue

types and mucins. Times required for each step are flexible in both the staining procedures

discussed here and in the previous embedding protocol. Different tissue types may require

different times. All tissue baths, especially xylene and ethanol ones, should be changed

frequently. Slides should not be allowed to dry during transfers. Solutions to common

embedding, sectioning, and staining problems are discussed in Preece (1972) and most other

manuals of histological technique.

Sections were deparaffinized and hydrated using a xylene-ethanol series (Table 2). Following

hydration, slides were stained in the pentachrome series, dehydrated in a series of acetic acid

dips followed by acetone, cleared in xylene and mounted in Permount (Table 2).

5. ANALYSIS

Each slide was examined microscopically to determine sex and stage of gonadal development. A

histopathological examination can also be made at this time, if desired (see Ellis et al., this

volume). Careful examination of early developmental stages may be needed to positively

distinguish males or females in early stages of development from individuals as yet

undifferentiated. Occasional hermaphrodites will also be found (all target species normally have

separate sexes). The stage in the gametogenic cycle was assigned based on the maturity of the

follicles and gametes and a numerical value assigned as described in Tables 3 and 4.

For mytilids and zebra mussels, a mean Gonadal Index can be determined from the sum of the

individual stage numbers. The index can vary from zero, if the entire population was spent or

resting, to five, if the population was fully ripe.

Cases of renewed gonadal development following spawning (stage 7) were common in oysters,

particularly along the Gulf of Mexico coast. These animals typically have a few remaining

large, mature ova and many developing ova that would normally be found in stages 3 or 4.

Accordingly, for oysters, further data reduction can better be achieved by comparing the

number of individuals with substantial gonadal development with those having little gonadal

volume using an egg/eggless ratio, calculated as

= 

the number of individuals at stages 3, 4, 5, 6 and 7

the number of individuals at stages 1, 2 and 8

223
Table 2. Tissue staining sequence.

Deparaffinization Phosphotungstic acid 2 min

xylene 5 min Orange G/Chromotrope stain 1.5 min

xylene 5 min Running tap water* 5 min

xylene 5 min Phosphomolybdic acid 2 min

100% ethanol 3 min Fast Green/Aniline Blue stain 3 min

100% ethanol 2 min

Dehydration

Hydration 1% acetic acid 20-25 dips

95% ethanol 2 min 1% acetic acid 20-25 dips

10% ethanol 2 min 1% acetic acid 20-25 dips

distilled water 2 min 1% acid acetone 20-25 dips

1% acid acetone 20-25 dips

Staining series 1% acid acetone 20-25 dips

Ferric Alum Mordant 6 min

Groat/Weigert Hematoxylin 30-45 min Clearing

Running tap water 3 min xylene 5 min

Basic ethanol 15 sec xylene 5 min

Running tap water 2 min

Acid Fuchsin stain 1.5 min Mounting

Running tap water* 5 min Mounting in Permount 24 hr to dry

* At these steps, no stain should remain between the slides and holding grooves in the slide rack

Table 3. Oyster development stages.

Developmental Stage Value Description

Sexually undifferentiated 1 Little or no gonadal tissue visible

Early development 2 Follicles beginning to expand

Mid-development 3 Follicles expanded and beginning to coalesce; no mature

gametes present

Late development 4 Follicles greatly expanded, and coalesced, but

considerable connective tissue remaining; some

mature gametes present

Fully developed 5 Most gametes mature; little connective tissue

remaining

Spawning 6 Gametes visible in gonoducts

Spawned 7 Reduced number of gametes; some mature gametes

still remaining; evidence of renewed reproductive

activity

Spawned 8 Few or no gametes visible; gonadal tissue atrophying

224
Table 4. Mussel and zebra mussel development stages.

Reproductive stage Description

Resting/spent gonad

Stage 0 Inactive or undifferentiated

Developing gonad

Stage 1 Gametogenesis has begun; no ripe gametes visible

Stage 2 Ripe gametes present; gonad developed to about one-third of its final size

Stage 3 Gonad increased in mass to about half the fully ripe condition; each follicle

contains, in area, about equal proportions of ripe and developing gametes

Stage 4 Gametogenesis still progressing, follicles contain mainly ripe gametes

Ripe gonad

Stage 5 Gonad fully ripe, early stages of gametogenesis rare; follicles distended

with ripe gametes; ova compacted into polygonal configurations; sperm

with visible tails

Spawning gonad

Stage 4 Active emission has begun; sperm density reduced; ova rounded off as

pressure within follicles is reduced

Stage 3 Gonad about half empty

Stage 2 Gonadal area reduced; follicles about one-third full of ripe gametes

Stage 1 Only residual gametes remain; some may be undergoing cytolysis

6. CONCLUSIONS

The procedures describe provide a semiquantitative ranking of reproductive stage but no

quantification of the amount of gametic tissue present. The strengths of this approach are that

it provides an assessment of sexual stage in the gametogenic cycle, and, if desired, allows for

a concomitant histopathological analysis, with a single sample preparation protocol. The

procedure cannot be performed on pooled samples, thus necessitating individual analysis of a

fraction of the samples pooled for other purposes or analysis of separate individuals. Thus, a

direct correspondence between, for example, hydrocarbon body burden and stage in the

gametogenic cycle may be difficult, because subsampling of individual animals will result in a

certain degree of bias in the measurement of contaminant body burden, normally around 10% in

adult oysters (Sericano et al., 1993) and more for smaller species. This bias, therefore, will

be size-, contaminant-, and time of year dependent.

If a quantitative gonadal/somatic index is desired, the technique of Choi and Powell (1993)

should be used. The latter technique measures the concentration of egg protein present,

however, it is not compatible with a concomitant histopathological analysis in that the standard

histological preparation for assessing reproductive stage is not used in the quantitative

analysis and tissue subsampling for histology cannot be done on the same individuals to be

analyzed quantitatively for gonadal-somatic index. Choi et al. (1993) have further discussed

the relative value of various approaches to gonadal evaluation. Overall, these authors found

that the same general trends could be identified using either the semiquantitative or

quantitative technique in most species, because normally an increase in gonadal volume occurs

225
more or less simultaneously with advancement in reproductive stage and because gamete

maturation occurs more or less evenly throughout the entire gonad.

7. REFERENCES

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Borcherding, J. (1990) Die Reproduktionsleistungen der Wandermuschel Dreissena polymorpha.

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Mo, C., and B. Neilson (1993) Weight and salinity effects on zinc uptake and accumulation for

the American oyster (Crassostrea virginica Gmelin). Environ. Pollut., 82:191-6.

Morales-Alamo, R., and R. Mann (1989) Anatomical features in histological sections of

Crassostrea virginica (Gmelin, 1791) as an aid in measurements of gonad area for

reproductive assessment. J. Shellfish Res., 8:71-82.

Newell, R. I. E. (1989) Species profiles: life histories and environmental requirements of

coastal fishes and invertebrates (North and Mid-Atlantic) - blue mussel. U.S. Fish. Wildl. Serv.

Biol. Rep. 82(11.102). U.S. Army Corps of Engineers, TR E1-82-4. 25 pp.

Powell, E. N., E. A. Wilson-Ormond and K-S. Choi (1993) Gonadal analysis - Crassostrea

virginica. In: Sampling and analytical methods of the National Status and Trends Program

National Benthic Surveillance and Mussel Watch Projects 1984-1992. Vol. II Comprehensive

descriptions of complementary measurements. NOAA Tech. Mem. NOS ORCA 71.

NOAA/NOS/ORCA, Silver Spring, MD. 55-62.

Preece, A. (1972) A Manual for Histologic Technicians. Little, Brown, and Company, Boston.

428 pp.

Seed, R. (1975) Reproduction in Mytilus (Mollusca: Bivalvia) in European waters. Pubblicazioni

della Stazione Zoologica di Napoli (Milan), 39(Suppl. 1):317-334.

Seed, R. (1976) Ecology. In: Marine Mussels: Their Ecology and Physiology. B. L. Bayne (ed.).

Cambridge University Press, New York, NY. 13-66.

Seed, R., and T. H. Suchanek (1992) Population and community ecology of Mytilus. In: The

Mussel Mytilus: Ecology, Physiology, Genetics and Culture. E. Gosling (Ed.). Developments in

Aquaculture and Fisheries Science, 25:87-169.

Sericano, J. L., T. L. Wade, E. N. Powell, and J. M. Brooks (1993) Concurrent chemical and

histological analyses: are they compatible? Chem. Ecol., 8:41-7.

Sprung, M. (1991) Costs of reproduction: a study on metabolic requirements of the gonads and

fecundity of the bivalve Dreissena polymorpha. Malacologia, 33:63-70.

Wilson, E. A., E. N. Powell, M. A. Craig, T. L. Wade, and J. M. Brooks (1990) The distribution of

Perkinsus marinus in Gulf coast oysters: its relationship with temperature, reproduction, and

pollutant body burden. Int. Rev. Gesamten Hydrobiol., 75:533-50.

Wilson, E. A., E. N. Powell, T. L. Wade, R. J. Taylor, B. J. Presley and J. M. Brooks (1992)

Spatial and temporal distributions of contaminant body burden and disease in Gulf of Mexico

oyster populations: the role of local and large-scale climatic controls. Helgol. Meeresunters.,

46:201-35.

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Perkinsus marinus Assay

E. N. Powell and M. S. Ellis

Haskin Shellfish Research Laboratory

Rutgers University

6959 Miller Ave.

Port Norris, NJ

USA

ABSTRACT

This document describes the procedures followed for the assay of Perkinsus marinus

prevalence and infection intensity in oyster specimens collected on the East and Gulf

Coasts of the US and in Puerto Rico by Rutgers University for the National Oceanic and

Atmospheric Administration's National Status and Trends Program Mussel Watch

Project.

1. INTRODUCTION

The determination of the health of oyster populations requires an analysis of Perkinsus

marinus prevalence and infection intensity. P. marinus is the most serious disease-producing

pathogen of East and Gulf coast oysters. This standard operating procedure is the accepted best

method for quantitating the presence of this parasite when an approximate or exact

quantification of disease intensity is required. A tissue homogenate or a section of mantle

tissue is incubated in thioglycollate medium for 14 days according to the method of Ray (1966).

A semiquantitative (Craig et al., 1989) or quantitative (Choi et al., 1989) assessment of

hypnospore number is then made microscopically.

2. EQUIPMENT AND SUPPLIES

2.1. Reagents

2.1.1. Chemicals

Chloramphenicol (chloromycetin), (C11H12C12N2O5) [56-75-7], 1 g lyophilized powder vials;

only available from veterinary supply houses.

Fluid thioglycollate medium, powder, T9032. Sigma Chemical Company, St. Louis, MO.

Iodine, (I2) [7553-56-2], 13380, purity 99%. Sigma Chemical Company, St. Louis, MO.

Nystatin (mycostatin) (C47H75NO17) [1400-61-9], 500,000 unit lyophilized powder, N3503.

Sigma Chemical Company, St. Louis, MO.

Potassium iodide (KI) [7681-11-0], P8256. Sigma Chemical Company, St. Louis, MO.

Potassium phosphate, monobasic, (KH2PO4) [7778-77-0], purity 99+%, P5379. Sigma

Chemical Company, St. Louis, MO.

Sodium chloride (NaCl) [7647-14-5], Purity >99.5%, S9625. Sigma Chemical Company, St.

Louis, MO.

Sodium hydroxide (NaOH) [1310-73-2], S5881. Sigma Chemical Company, St. Louis, MO.

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2.1.2. Solutions

2.1.2.1. Thioglycollate medium preparation

A mixture of 20 g NaCl, 29.3 g dehydrated fluid thioglycollate medium, and 1 L distilled water

was heated while stirring until the medium dissolved and the solution became a transparent

golden-yellow color. After cooling, the solution was dispensed, 10 mL at a time, into 18-mL

culture tubes which were subsequently autoclaved and sealed.

Thioglycollate maintains anaerobic conditions in the culture tube as well as providing needed

nutrients and an appropriate osmotic environment. Therefore, tubes were sealed tightly and

opened only briefly for injection of antibiotic and addition of tissue as subsequently described.

After autoclaving, prepared tubes were stored in the dark until use and then returned

immediately to the dark for tissue incubation. Unused, autoclaved tubes of medium can be

stored for many months in the dark without deterioration. Occasionally, tubes became cloudy

or the medium congealed. In both cases, these individual tubes were discarded. However, the

remaining tubes in a batch remained usable.

2.1.2.2. Antibiotic solution

Five milliliters of sterile water were added to a 1-g vial of chloromycetin (chloramphenicol)

sterile powder using a sterile needle and syringe. Ten milliliters of sterile water were added to

a 500,000-unit vial of mycostatin (nystatin) powder with a sterile needle and syringe. Both

vials were shaken to dissolve the solids. Using a sterile needle and syringe, 2.5 mL of

chloromycetin solution were added to the mycostatin vial, the mixture shaken, and

refrigerated until use. The solution can be stored safely for several weeks if refrigerated. The

remaining chloromycetin may be used in a second mycostatin vial.

2.1.2.3. Lugol's iodine solution

Four grams of potassium iodide and 2 g of iodine crystals were dissolved in 100 mL of distilled

water, allowed to stand 24 hr, and filtered. The solution was stored in a dark bottle. The

solution remained stable for many weeks.

2.1.2.4. PBS(II)

Enough NaCl and KH2PO4 are dissolved in distilled water to produce a 0.15 M NaCl and 0.01 M

KH2PO4 solution. The pH is adjusted to 7.4 with NaOH or HCl as required.

2.2. Equipment

Autoclave

Automatic pipette or repipettes, 50 μL and 200 mL capacity, 13-707-30. Fisher Scientific,

Pittsburgh, PA.

Brinkmann polytron tissue homogenizer, Brinkmann Instrument Co., Westbury, NY.

Heater/stirrer, Corning, 11-495-52C. Fisher Scientific, Pittsburgh, PA.

Microscope, Wild dissecting. Fisher Equivalent Stereomaster Zoom, 12598-14. Fisher

Scientific, Pittsburgh, PA.

Repipettes, 10-50 mL adjustable volume, 13-688-70. Fisher Scientific, Pittsburgh, PA.

Slide warmer, 12-594. Fisher Scientific, Pittsburgh, PA.

Vortex mixer, Vortex-Genie 2 mixer, 12-812. Fisher Scientific, Pittsburgh, PA.

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3. TISSUE COLLECTION

Twelve large oysters were collected from each field location. Analysis of twelve oysters was

sufficient to determine prevalence and infection intensity. The Mussel Watch sampling protocol

includes obtaining specimens from three separate locations at each primary site. Comparisons

of replicate samples have consistently shown significant differences no more frequently than

expected by chance (Craig et al., 1989; Wilson et al., 1990). Thus four oysters were taken

from each of the three sampling locations per site to obtain the twelve oysters for analysis.

Oysters were immediately put on ice, but not allowed to be submerged in fresh water by

allowing meltwater to drain continuously from the containers, and returned to the laboratory

where they were assayed within 24 hr of collection.

Each oyster was opened with an oyster knife. Maximum anterior-posterior length, as defined

by Morales-Alamo and Mann (1989), was recorded by visual inspection. Using sterile

dissecting scissors and forceps, a small piece of approximately 1 cm2 of mantle tissue was

excised from the lower valve just posterior to the palps. The tissue was placed in a culture

tube containing the thioglycollate medium to which 50 μL of chloramphenicol-nystatin antibiotic

solution was added.*

The tube was immediately recapped and shaken by inversion several times to assure that the

tissue was submerged in the medium. When processing many samples, it was convenient to add

the antibiotic solution to the vials prior to, but not earlier than 24 hr before, opening the

oysters. The culture tubes were placed in the dark at room temperature and incubated for 14

days. At the end of 14 days, the tissue was analyzed or, if inconvenient, the tube was placed in

a refrigerator in the dark. Tissues ready to be analyzed can be kept for at least 3 months

without deterioration if the culture tubes are kept dark and refrigerated.

4. TISSUE ANALYSIS

4.1. Semiquantitative method

After the incubation period, the oyster tissue was removed from the culture tube using a

sterile needle and placed on a microscope slide. The tissue sample was teased apart using

sterile needles to assure even staining with Lugol's iodine solution. The tissue was flooded with

Lugol's solution using a Pasteur pipette, covered with a cover slip and examined

microscopically. P. marinus hypnospores appear as black spheres of 10 to 200 μm diameter

when viewed through a dissecting microscope. Usually, hypnospores exceed 40 μm in size,

however, occasionally hypnospores develop without enlargement. An infection intensity was

assigned to each tissue sample based on the number or coverage of enlarged P. marinus

hypnospores observed in the tissue using the scale in Table 1. Note that the scale ranks tissue

by the number or coverage of hypnospores after enlargement. Accordingly, those tissues

where enlargement occasionally fails to occur must be ranked on Mackin's scale as if the cells

were enlarged.

*

 Ray (1966) discusses the use of various antibiotics. Although certain laboratories use alternative antibiotics, both Ray

(1966) and Quick and Mackin (1971) concluded that the recommended combination of nystatin and chloramphenicol was

superior.

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Table 1. Semiquantitative scale of infection intensity for Perkinsus marinus [adapted from

Mackin (1962) by Craig et al. (1989)].

Letter Infection Numerical Description

designation intensity value

N Negative 0.00 No hypnospores present

VL Very light 0.33 1-10 hypnospores

L- 0.67 11-74 hypnospores

L Light 1.00 75-125 hypnospores

L+ 1.33 >125 hypnospores but much less than 25% of

tissue is hypnospores

LM- 1.67 <25% of tissue is hypnospores

LM Light/moderate 2.00 25% of tissue is hypnospores

LM+ 2.33 >25% but much less than 50% of tissue is

hypnospores

M- 2.67 >25% but <50% of tissue is hypnospores

M Moderate 3.00 50% of tissue is hypnospores

M+ 3.33 >50% but much less than 75% of tissue is

hypnospores

MH- 3.67 >50% but <75% of tissue is hypnospores

MH Moderately heavy 4.00 75% of tissue is hypnospores

MH+ 4.33 >75% but much less than 100% of tissue is

hypnospores

H- Heavy 4.67 >75% of tissue is hypnospores but some oyster

tissue is still visible

H 5.00 Nearly 100% of tissue is hypnospores

The technique depends on the assumption discussed by Ray (1954) that hypnospores develop

from single P. marinus cells without replication and that all P. marinus cells develop into

hypnospores. Gauthier and Fisher (1990) discuss an adaptation of this method using oyster

hemolymph which can be used to non-destructively follow the progression of P. marinus

infection.

To maintain quality control, blind assays were conducted among the slide readers to correct

for any technician bias that may be present with any semiquantitative technique. We encourage

other users to standardize their analyses with laboratories already using the technique so that

data are comparable.

4.2. Quantitative method

The assessment of infection intensity using a piece of mantle tissue is rapid, inexpensive, and

can be done with little tissue loss on animals destined for body burden analysis, permitting a

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direct comparison. However, use of a single tissue section introduces three potentially

significant inaccuracies.

The single-tissue section method does not quantitatively assess cell number although an

approximate conversion to cell number can be used (Section 5).

Tissue-to-tissue heterogeneity in infection intensity was typically as large as two levels on

Mackin's scale (Table 1) (e.g., from L- to L+).

False negatives were frequently encountered at low infection levels. A lethal infection from a

single infective cell required on the order of 22 to 28 doublings of the P. marinus population.

The first 10 to 12 were likely to be read as negative because cell density was low enough that

a piece of mantle tissue examined may easily not contain any cells. This error reduced the

value of prevalence as a primary determinant of health because the method used was

particularly poor at identifying truly uninfected oysters.

A quantitative method measuring the total body burden of parasites resolves these three

inaccuracies but requires substantially more technician time and, obviously, the use of entire

animals.

After shucking, the oyster meat was homogenized using a Brinkmann Polytron tissue

homogenizer at level 3 (moderately-low speed) for 2 min. The homogenized oyster tissue was

incubated in thioglycollate medium as described previously. After 14 days, the volume of the

mixture in each flask was measured using a graduated cylinder. The mixture was poured back

into the original flask and stirred vigorously. A 30-mL subsample was immediately removed

and placed into a 50-mL tube. The subsample was centrifuged at 6000 rpm for 10 min and the

supernatant discarded. Approximately 30 mL of 2 M NaOH were added to each pellet and the

mixture incubated at 50 °C in a water bath for 1 hr. After incubation, the sample was

centrifuged at 6000 rpm for 15 min and the supernatant was removed. The pellet was

resuspended in 30 mL of phosphate buffered saline [PBS(II)] and centrifuged using the same

conditions. The pellet was washed two additional times. The pellet volume was measured. This

required the addition of some PBS(II) in some instances. The suspension was stirred using a

Vortex mixer. Aliquots of 100 μL were taken and 100 μL of Lugol's solution was added to each.

The number of hypnospores was counted in at least 10 100-μl aliquots from each sample using

a hemacytometer and the mean number of hypnospores reported.

5. CALCULATIONS

Data were reported as prevalence (percent infected) and mean or median infection intensity

calculated using the semiquantitative scale in Table 1. The calculation of median infection was

frequently desirable because the semiquantitative scale used was truncated at both extremes

and infection intensity in a sampled population was often not normally distributed. In particular,

a few relatively heavily infected individuals were frequently encountered in populations

characterized by low overall infection intensities, and uninfected individuals could be collected

in most populations at certain times of the year because transmission rates are sufficiently

slow. If the quantitative scale was desired, data were reported as number of P. marinus cells

per gram wet weight of oyster tissue and then converted into the semiquantitative equivalent

using the formula:

 Fisher and Oliver (1996) have recently described a modification of this method.

232
number of hypnospores = 1409.9 (100.64296x)

where x is the semiquantitative designation (Choi et al., 1989).

6. CONCLUSIONS

The described technique provides a quantitative or semi-quantitative method to determine the

presence and infection intensity of Perkinsus marinus in oysters. South of Long Island Sound,

Perkinsus marinus analysis is a good indicator of population health because P. marinus is a

principal cause of mortality in most populations. The thioglycollate method is considerable more

accurate than examination of tissue sections in routine histopathological analysis, and thus

should be a standard component of histopathological analysis for determining population health.

7. REFERENCES

Choi, K-S., E. A. Wilson, D. H. Lewis, E. N. Powell, and S. M. Ray (1989) The energetic cost of

Perkinsus marinus parasitism in oysters: quantification of the thioglycollate method. J.

Shellfish Res., 8: 125-31.

Craig, M. A., E. N. Powell, R. R. Fay, and J. M. Brooks (1989) Distribution of Perkinsus

marinus in Gulf coast oyster populations. Estuaries, 12: 82-91.

Fisher, W. S., and L. M. Oliver (1996) A whole-oyster procedure for diagnosis of Perkinsus

marinus disease using Ray's fluid thioglycollate culture medium. J. Shellfish Res., 15:109-17.

Gauthier, J. D., and W. S. Fisher (1990) Hemolymph assay for diagnosis of Perkinsus marinus

in the oyster Crassostrea virginica (Gmelin, 1791). J. Shellfish Res., 9:367-71.

Morales-Alamo, R., and R. Mann (1989) Anatomical features in histological sections of

Crassostrea virginica (Gmelin, 1791) as an aid in measurements of gonad area for reproductive

assessment. J. Shellfish Res., 8:71-82.

Mackin, J. G. (1962) Oyster disease caused by Dermocystidium marinus and other

microorganisms in Louisiana. Publ. Inst. Mar. Sci. Univ. Texas, 7:132-229.

Quick,. J. A., and J. G. Mackin (1971) Oyster parasitism by Labyrinthomyxa marina in Florida.

Fl. Dept. Nat. Resour. Mar. Res. Lab. Prof. Pap. Ser., 13:1-55.

Ray, S. M. (1954) Biological studies of Dermocystidium marinus, a fungus parasite of oysters.

Rice Inst. Pamph. Monogr. Biol. Special Issue, 114 pp. Rice University, Houston, TX.

Ray, S. M. (1966) A review of the culture method for detecting Dermocystidium marinus, with

suggested modifications and precautions. Proc. Natl. Shellfish. Assoc., 54: 55-69.

Wilson, E. A.. E. N. Powell, M. A Craig, T. L. Wade, and J. M. Brooks (1990) The distribution of

Perkinsus marinus in Gulf coast oysters: its relationship with temperature, reproduction, and

pollutant body burden. Int. Rev. Gesamten Hydrobiol., 75:533-50.

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234