ABSTRACT
ORMOND, ROBERT BRYAN. Advancement in the Man-In-Simulant-Test Methodology and Development of Next Generation Manikin for Chemical and Biological Protection Research. (Under the direction of Dr. Roger L. Barker and Dr. Keith R. Beck).
The Man-In-Simulant-Test (MIST) has been the primary method used to evaluate full ensembles with regards to their chemical protective performance since it was developed by the U.S.
Army during the early 1990s. MIST evaluations involve exposing human test subjects wearing full protective ensembles to a low toxicity simulant for chemical warfare agents. The target property that is assessed by the MIST is the ensemble’s ability to prohibit or minimize inward leakage of the simulant through the closures and interfaces such as seams, seals, or zippers.
The first area that was addressed by this research was the advancement of the current MIST methodology. A detailed extraction and analysis method was developed to remove the simulant, methyl salicylate (MeS), from the Passive Adsorbent Dosimeters (PADs) that are used to measure the leakage inside the ensemble. The removal of MeS from the adsorbent was shown to have an extraction efficiency greater than 98% across the entire expected range of MeS amounts. The liquid chromatography method was shown to be capable of detecting MeS in the expected ranges, provided an overall limit of quantitation of 30 ng/PAD, and only required 4.5 minutes to analyze a single sample.
A bench-scale MIST chamber was developed to quickly expose multiple PADs or materials at various MeS concentrations. The chamber was shown to be capable of maintaining a desired concentration for an extended period of time and could also be repeatedly filled to the same conditions across multiple days.
An extensive investigation was conducted to characterize the PADs based on the consistency of their physical properties and the effect of varying the exposure conditions on the measured uptake rate. Within a single lot, the PADs were shown to be relatively consistent in surface area and responded linearly to a range of exposure dosages. A significant variability between PAD lots was
observed and should be further investigated. The PAD handling procedures were also investigated,
and it is recommended to either extract the PADs within an hour of being exposed or store them at
extremely cold temperatures to prohibit any mass transfer from occurring during storage.
Various fabrics were investigated to determine their MeS uptake rates in both the bench-scale and full-scale MIST chambers. Fabrics comprised of 100 % polyester or cotton were shown to adsorb very little MeS, but incorporating only 5% spandex greatly increased both the capacity and uptake rate of the fabrics. A 95/5 polyester/spandex jersey fabric was found to have a thirty minute uptake rate very similar to the values reported for human skin, and therefore could potentially serve as a skin simulant in MIST protocols utilizing manikins.
The second main area that was addressed by this research was the development of a next generation manikin for use in MIST evaluations. An articulated thermal manikin was attached to an elliptical walker to increase the range of motion. A baseline for the observed variability in MIST results with human subjects was determined by conducting three separate trials with eight human subjects in the MIST Facility at NC State University. These results were compared to three manikin evaluations in the same NFPA 1994 Class 3 Ensemble. It was found that manikins and human subjects can produce consistent MIST results. It was also confirmed that three manikin evaluations produce the just as much variability as three human subjects, and therefore should not be used to replace a standard eight human subject test.
Advancement in the Man-In-Simulant-Test Methodology and Development of Next Generation Manikin for Chemical and Biological Protection Research
by Robert Bryan Ormond
A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy
Fiber and Polymer Science
Raleigh, North Carolina
2012
APPROVED BY:
______
Donald B. Thompson Ronald Baynes Committee Member Minor Committee Member
______
Roger L. Barker Keith R. Beck Chair of Advisory Committee Co-Chair of Advisory Committee
DEDICATION
First and foremost, this dissertation is dedicated to the memory of all the victims of the terrorist attacks on September 11, 2001, and to the soldiers, policemen, firefighters, and other first responders
that put their lives in harm’s way each and every day to protect complete strangers.
This dissertation is also dedicated to:
• To my Grandfather, Ray Maready, for teaching me that in life, just as in checkers, if you don’t
make a move you will get blown off the board, and also for showing me that no matter what odds
you are given or what adversity you face, you must stay strong in your faith and never give up.
• To my Grandmother, Louise, for showing me how to put others first and always take care of my
family, and also for teaching me how to make one of the best pizzas you will ever taste.
• To my Aunt, Yvonne, and her family for reminding me to always be prepared because you never
know when you will need your electrical tape.
• To my Uncle Phil, Aunt Angel, and their family for allowing me to experience the world of music
and also the world of college football and basketball through a trumpet.
• To my sisters, Lisa and Heather, for showing me what true inner strength looks like and how to
keep going everyday no matter what stands in your way.
• To my nieces, Hailey, Kylie, and Lily, and my nephews, Ethan and Josh, for letting me know
how it feels to have someone look up to you.
• To my mother, Juanita, for teaching me how to be compassionate, patient, and for molding me
into the man that I have become.
ii • To my father, Robert, for showing me how to stand firm in my beliefs and never compromise
myself to satisfy other people.
• To my wife, Sasha, for supporting me, listening when I needed to vent, and for allowing me to do
the same for you as we have traveled this road side by side.
• To my best friends, my boxer/bulldogs, Cotton and Chroma, who never cared what went right or
wrong during the day or what I did or didn’t finish, but always met me at the door only expecting
a scratch behind the ears.
• Finally, to my entire family for believing in me and encouraging me to never give up and always
push forward. I would be a shell of myself without the impact that each and every one of you
have had on my life.
iii BIOGRAPHY
Robert Bryan Ormond was born to Robert and Juanita Ormond on December 12, 1984 in
Williamston, NC. With his father being a minister in the Pentecostal Holiness Church, most of his childhood was spent in various parts of eastern North Carolina. He was raised alongside his two older sisters, Lisa and Heather. Bryan graduated as valedictorian of North Johnston High School in
Kenly, NC in May of 2003.
Following graduation, Bryan moved to Raleigh, NC to attend NC State University’s College of Textiles. As a student, Bryan was heavily involved with many organizations both in the College of
Textiles and across the university. He spent two seasons playing trumpet for the Power Sound of the
South Marching Band and four seasons as a member of the Wolfpack Basketball Pep Band. Bryan also served as the President of both the Phi Psi National Textile Fraternity and NCSU’s Student
Chapter of the AATCC. When not engrossed in his studies, Bryan also worked as an intern at Cotton
Incorporated for over three years. In 2007, Bryan graduated Summa Cum Laude with a B.S. in
Polymer and Color Chemistry with an American Chemical Society certification. He was named the
2007 AATCC Outstanding College Graduate of the Year and also received both the College of
Textiles Leadership Award and the Phi Psi Senior Award for his dedication to the college.
After completion of his undergraduate degree, Bryan began pursuing his doctorate at the
Center for Research on Textile Protection and Comfort at NC State. Bryan has presented his research
on the Man-In-Simulant-Test at multiple conferences both domestic and international and will
graduate with a PhD in Fiber and Polymer Science in May 2012.
Bryan and his wife, Sasha, were married in August 2008 and now reside in Raleigh, NC with
their two boxer/bulldogs, Cotton and Chroma.
iv ACKNOWLEDGMENTS
I would like to express my appreciation and gratitude to members of my research committee
for the guidance that they provided over the past five years. I would like to thank Dr. Roger Barker
for serving as my research advisor, allowing me to take my research in the direction that I believed it
should go, and pushing me to become an expert in my field. I would also like to specifically thank
Dr. Keith Beck for allowing me to learn from his extensive knowledge of analytical chemistry. The experience and techniques that I gained from the time spent with him will be invaluable as I progress into my future career.
I would also like to acknowledge and thank all of the current and former members of the
TPACC family for the support and guidance that they provided. I am grateful to Shawn Deaton, Don
Thompson, Gail Liston, John Morton-Aslanis, Mark Martin, Kevin Ross, and Michelle Lucas. I am also thankful for my fellow graduate students, Lee Gladish, Ashley Bradham, Taylor O’Cain, Andrew
White, Lauren Deuser, Jessica Watkins, and Marika Walker, for the countless hours that they put in to help conduct the MIST trials. I am also grateful for my fellow doctoral student, Dr. Alex Hummel, for the mental support that we were able to provide each other during this long and arduous process.
I would like to thank RDECOM for providing the financial support for the project and to acknowledge the work of Hai Bui and Brian Davis in the College of Textiles’ machine shop and weaving lab for providing their services to my project. I would also like to express my appreciation to the City of Raleigh firefighters that served as subjects for all of the human subject testing.
Finally, I would like to express my most sincere appreciation to my wife, Sasha, and my entire family for the sacrifices that were made to get me to this point and for supporting me and pushing me forward when I could not do it on my own.
v TABLE OF CONTENTS
LIST OF TABLES ...... xii
LIST OF FIGURES ...... xv
LIST OF EQUATIONS ...... xx
LIST OF ABBREVIATIONS ...... xxi
CHAPTER 1: Introduction and Purpose of Research ...... 1 1.1 Purpose of Research ...... 1 1.2 Research Objectives ...... 2 1.2.1 Advancement of the MIST Methodology ...... 2 1.2.2 Development of Next Generation Manikin for Chemical and Biological Protection Research ...... 2
CHAPTER 2: Chemical Warfare: History, Agents, and Toxicology ...... 4 2.1 Introduction to the Modern Age of Chemical Warfare ...... 4 2.1.1 Chemical Warfare in the Ancient World ...... 4 2.1.2 Dr. Fritz Haber: Feeder of Nations and Father of Chemical Warfare ...... 5 2.1.3 The Relationship between the Textile Industry and Chemical Warfare ...... 6 2.2 The First Generation of Chemical Warfare Agent: Irritants, Chlorine, and Phosgene ...... 8 2.2.1 The First Large Scale Use of Chlorine as a Chemical Warfare Agent ...... 8 2.2.2 Chlorine: Mechanism of Action ...... 10 2.2.3 Protection Against Chlorine: First Respirators Used in Warfare ...... 10 2.2.4 Development of More Effective Respirators and More Lethal Agents ...... 12 2.3 The Second Generation of Chemical Warfare Agents: Vesicants ...... 19 2.3.1 Unveiling of Vesicant Agents to the Battlefield ...... 19 2.3.2 Sulfur Mustard: Physical Properties and Effectiveness of Use ...... 20 2.3.3 Additional Vesicant Agents ...... 25 2.3.4 Basics Principles of Percutaneous Absorption ...... 26
vi 2.3.5 Vesicants: Mechanism of Action and Toxicity ...... 32 2.4 The Third Generation of Chemical Warfare Agents: Nerve Agents ...... 39 2.4.1 Development of Organophosphorous Nerve Agents ...... 39 2.4.2 Nerve Agents: Physical and Chemical Properties ...... 42 2.4.3 Nerve Agents: Mechanism of Action ...... 44
CHAPTER 3: Modern Chemical Protective Ensembles ...... 48 3.1 Introduction to Modern Chemical Protective Equipment ...... 48 3.1.1 Need for Personal Protective Equipment for Specific Routes of Exposure ...... 48 3.2 Modern Respiratory Protection ...... 49 3.2.1 Overview of Different Levels and Types of Respiratory Protection...... 49 3.3 Modern Total Body Protection...... 58 3.3.1 Guidelines for Selection of Appropriate Level of Chemical Protection ...... 58 3.3.2 Performance Criteria for Chemical Protective Ensembles ...... 63 3.4 Standard Test Methods for Evaluating Chemical Protective Materials ...... 72 3.4.1 Introduction to Material Level Chemical Testing ...... 72 3.4.2 Chemical Permeation Test Method and Battery of Chemicals ...... 73 3.4.3 Test Methods for Resistance to Chemical Penetration...... 75
CHAPTER 4: Man-In-Simulant-Test: Background, Protocols, and Analysis ...... 77 4.1 Introduction to Man-In-Simulant-Test (MIST) ...... 77 4.1.1 Background and Basic MIST Methodology ...... 77 4.2 Theories and Principles of MIST Methodology ...... 80 4.2.1 Methyl Salicylate as a Chemical Warfare Agent Simulant ...... 80 4.2.2 Principles of Passive Diffusion and Passive Adsorbent Dosimeters ...... 83 4.3 Differences between ASTM F 2588 and TOP 10-2-022 Methods ...... 87 4.3.1 Differences between the Environmental Conditions ...... 87 4.3.2 Differences between the Exercise Protocols ...... 88 4.3.3 Differences between the PAD Placement ...... 90 4.4 Development and Explanation of Body Region Hazard Analysis ...... 92
vii 4.4.1 Development of Model to Predict Systemic Effects from Nerve Agent VX Exposure...... 92 4.4.2 Development of Model to Predict Localized Effects from Sulfur Mustard Exposure ...... 95
CHAPTER 5: Development of Methods for Extraction and Analysis of Methyl Salicylate in Passive Adsorbent Dosimeters ...... 100 5.1 Introduction and Background ...... 100 5.2 Experimental Methods ...... 101 5.2.1 Extraction of Tenax ® from Passive Adsorbent Dosimeters ...... 101 5.2.2 Extraction of Methyl Salicylate from Tenax ® ...... 104 5.2.3 High Performance Liquid Chromatography (HPLC) Analysis Method ...... 105 5.2.4 Development of Calibration Curve for Methyl Salicylate ...... 105 5.2.5 Calculation of Method Detection Limit and Limit of Quantitation ...... 107 5.2.6 Determination of Variability in Solvent Volume Calculation ...... 108 5.2.7 Determination of Extraction Efficiency for Common Solvents ...... 109 5.2.8 Determination of Extraction Efficiency at Various Spiked Amounts ...... 109 5.3 Results and Discussion ...... 110 5.3.1 Extraction of Tenax® from Passive Adsorbent Dosimeter ...... 110 5.3.2 Development of Analytical Method and Calibration Curve for Methyl Salicylate ...... 110 5.3.3 Calculation of Limit of Detection and Limit of Quantification ...... 114 5.3.4 Determination of Variability in Solvent Volume Calculation ...... 116 5.3.5 Extraction Efficiency for Common Solvents ...... 118 5.3.6 Determination of Extraction Efficiency at Various Spiked Amounts ...... 119 5.4 Conclusions ...... 120
CHAPTER 6: Development of Bench-Scale Methyl Salicylate Exposure Chamber ...... 122 6.1 Introduction and Background ...... 122 6.2 Experimental Methods ...... 123 6.2.1 Design and Construction of Bench-Scale Chamber ...... 123 6.2.2 Methyl Salicylate Vapor Generation System ...... 125
viii 6.2.3 Methyl Salicylate Concentration Measurement ...... 127 6.2.4 Investigation into the Repeatability of Chamber Conditions ...... 129 6.2.5 PAD Exposure Methodology ...... 130 6.2.6 PAD Extraction Methodology...... 130 6.2.7 High Performance Liquid Chromatography (HPLC) Analysis Method ...... 131 6.3 Results and Discussion ...... 131 6.3.1 Consistency and Repeatability of Chamber Conditions ...... 131 6.3.2 Repeatability of PAD Exposures in Bench-Scale Chamber ...... 135 6.3.3 Comparison of Bench-Scale and Full-Scale MIST Chamber PAD Exposures ..... 135 6.4 Conclusions ...... 138
CHAPTER 7: Factors Influencing the Uptake Rate of Passive Adsorbent Dosimeters ...... 140 7.1 Introduction and Background ...... 140 7.2 Experimental Methods ...... 146 7.2.1 Measurement of the PAD Physical Dimensions ...... 146 7.2.2 Results from All PAD Exposures ...... 146 7.2.3 Effect of Challenge Concentration and Exposure Time on PAD Uptake Rate ..... 147 7.2.4 Effect of Post-Exposure Time on PAD Uptake Rate ...... 148 7.2.5 Effect of Storage Temperature on PAD Uptake Rate ...... 149 7.2.6 Lot-to-Lot Variability in PAD Dimensions and Uptake Rate ...... 149 7.2.7 PAD Extraction Methodology...... 149 7.2.8 Supplemental Extraction Methods for Desorption Tubes and Adhesive Film ...... 150 7.2.9 High Performance Liquid Chromatography (HPLC) Analysis Method ...... 150 7.3 Results and Discussion ...... 151 7.3.1 Variation in Physical Properties of the PADs ...... 151 7.3.2 Uptake Rate Variation with Challenge Concentration and Exposure Time ...... 154 7.3.3 Effect of Post-Exposure Storage Time and Temperature on the PAD Uptake Rate ...... 159 7.4 Conclusions ...... 163
ix CHAPTER 8: Investigation of Methyl Salicylate Uptake Rates of Fabrics ...... 166 8.1 Introduction and Background ...... 166 8.2 Experimental Methods ...... 168 8.2.1 Bench-Scale Exposures ...... 168 8.2.1.1 Bench-Scale Exposure Methodology ...... 168 8.2.1.2 Methyl Salicylate Extraction from PADs and Fabrics ...... 170 8.2.1.3 Gas Chromatography (GC) Analysis Method ...... 171 8.2.2 Full-Scale MIST Exposures ...... 172 8.2.2.1 Full-Scale Exposure Methodology ...... 172 8.2.2.2 Methyl Salicylate Extraction from Fabrics ...... 173 8.2.3 High Performance Liquid Chromatography (HPLC) Analysis Method ...... 173 8.3 Results and Discussion ...... 174 8.3.1 Bench-Scale Exposures ...... 174 8.3.2 Full-Scale Exposures ...... 177 8.4 Conclusions ...... 180
CHAPTER 9: Investigation into the Variability of Man-In-Simulant-Test Results with Human Subjects and Feasibility of Incorporating Articulated Manikins...... 181 9.1 Introduction ...... 181 9.2 Experimental Methods ...... 186 9.2.1 Methods Used to Adapt NEWTON Manikin for MIST Testing ...... 186 9.2.2 MIST Exposure Methodology...... 190 9.2.3 PAD Extraction Methodology...... 193 9.2.4 High Performance Liquid Chromatography (HPLC) Analysis Method ...... 194 9.3 Results and Discussion ...... 194 9.3.1 Variability in MIST Results from Multiple Human Subjects ...... 194 9.3.2 Feasibility of Using a Manikin in MIST Testing ...... 201 9.4 Conclusions ...... 204
x CHAPTER 10: Research Conclusions, Recommendations, and Proposed Future Research ...... 207 10.1 Summary of Research and Overall Conclusions ...... 207 10.2 Research Conclusions ...... 207 10.2.1 Conclusions Pertaining to the Advancement of the MIST Methodology ...... 207 10.2.2 Conclusions Pertaining to the Development of a MIST Manikin ...... 209 10.3 Proposed Future Research ...... 212 10.3.1 Future Research on the MIST Methodology ...... 212 10.3.2 Future Research on MIST Manikin Development and Incorporation ...... 212
REFERENCES ...... 214
APPENDIX ...... 221 APPENDIX A: Body Region Hazard Analysis ...... 222 A.1 Body Region Hazard Analysis for ASTM F2588 MIST Protocol ...... 222 A.1.1 Calculation of Local Physiological Protective Dosage Factors ...... 222 A.1.2 Calculation of Systemic Physiological Protective Dosage Factor ...... 226 A.2 Body Region Hazard Analysis for Military TOP 10-2-022 Protocol ...... 230 A.2.1 Calculation of Local and Systemic Protection Factors and Effects ...... 230
xi LIST OF TABLES
Table 2-1. Physical Properties and Chemical Structures for Common Vesicants ...... 22
Table 2-2. Ratio of Chemical Warfare Fatalities to Chemical Warfare Casualtie ...... 24
Table 2-3. Percentage of Body Surface Area for Anatomical Regions ...... 27
Table 2-4. Variation in Epidermal and Dermal Thickness between Races ...... 30
Table 2-5. Distribution of Mustard Gas Injuries on Bodies of World War I Casualties ...... 33
Table 2-6. Dose-Response Profiles by Route of Exposure to Sulfur Mustard at 16-27°C ...... 35
Table 2-7. Dose-Related Effects of Sulfur Mustard on Skin at 16-27°C ...... 36
Table 2-8. Dose-Related Effects of Sulfur Mustard on Eyes at 16-27°C ...... 37
Table 2-9. Dose-Related Effects of Sulfur Mustard on the Respiratory System at 16-27°C ...... 38
Table 2-10. Physical and Chemical Properties of Organophosphorous Nerve Agents ...... 43
Table 3-1. IDLH Values for Common Chemical Warfare Agents ...... 54
Table 3-2. EPA Levels of Chemical Protective Equipment ...... 60
Table 3-3. Overview of Key NFPA 1991 and 1992 Performance Criteria ...... 66
Table 3-4. Example of DuPont™ NFPA 1991 Vapor-Protective Ensemble ...... 67
Table 3-5. Example of DuPont™ NFPA 1992 Liquid Splash-Protective Ensemble ...... 68
Table 3-6. Overview of Key NFPA 1994 Performance Criteria ...... 69
Table 3-7. Example of DuPont™ NFPA 1994 Class 2 Ensemble ...... 70
Table 3-8. Various Chemical Protective Ensembles from Lion® Apparel and Blauer® ...... 71
Table 3-9. Concentration or Liquid Density of Permeation Test Chemicals...... 74
Table 4-1. Structures and Physical Properties of CWAs and MeS...... 82
xii Table 4-2. Differences in MIST Parameters for ASTM and TOP Standards ...... 88
Table 4-3. Differences in MIST Exercise Protocols for ASTM and TOP Standards ...... 89
Table 4-4. Median Effective Doses of VX by Body Region ...... 94
Table 4-5. Median Effective Doses for HD Vesication by Body Region ...... 96
Table 4-6. Probability (%) of Observing Effects for HD Exposure Range for the Specific Body Region ...... 98
Table 4-7. Estimated ECt10 and ECt50 Values for Severe Effects from HD Exposure ...... 99
Table 5-1. Methyl Salicylate Calibration Solutions for HPLC...... 106
Table 5-2. Method Detection Limit and Limit of Quantitation for Analytical Method ...... 115
Table 5-3. Method Detection Limit and Limit of Quantitation for PAD Analysis ...... 116
Table 5-4. Basic Statistical Analysis of Solvent Volumes ...... 118
Table 6-1. Bench-Scale Parameter Set Points ...... 129
Table 6-2. Calculated Concentration of MeS in Boiler Flow Stream ...... 134
Table 6-3. Basic Statistical Analysis of Bench-Scale Chamber Trials ...... 135
Table 6-4. Comparison of Basic Statistical Values for the Bench-Scale and Full-Scale Exposures .. 137
Table 7-1. Statistical Values for the Variation in the Calculated Uptake Rates. Data for “All Exposed PADs” are Averaged from 44 Total PADs at Each Concentration. Data for “PADs Exposed for 30+ Minutes” are Averaged from 28 total PADs at Each Concentration. ... 158
Table 8-1. Identification and Properties for Fabrics Used in Bench-Scale Exposures ...... 169
Table 8-2. Identification and Properties for Fabrics Used in Full-Scale MIST Exposures ...... 172
Table 9-1. Modified MIST Exercise Protocol for Human Subjects and Manikin ...... 190
Table 9-2. Ensembles Used in Human-Manikin MIST Comparisons ...... 193
Table A-1. Site Specific Onset of Symptoms Exposure Dosage (OSED) by PAD Location ...... 226
xiii Table A-2. ED50i Values by PAD and Body Location for Systemic PPDF Calculation ...... 227
Table A-3. PADs Mapped to Each Body Region According to TOP 10-2-022 ...... 231
xiv LIST OF FIGURES
Figure 2-1. British Black Veiling Respirator...... 12
Figure 2-2. British ‘Hypo’ Helmet ...... 13
Figure 2-3. Machine gunners wearing the PH Helmet ...... 15
Figure 2-4. Large Box Respirator (left) and Small Box Respirator (right ...... 16
Figure 2-5. German WWI Gas Mask (Gummimaske) ...... 18
Figure 2-6. British Livens Projector ...... 18
Figure 2-7. Anatomy of Human Skin ...... 28
Figure 2-8. Brick-and-Mortar Model of Dermal Absorption ...... 31
Figure 2-9. Mechanism of Acetylcholinesterase Activity, Inhibition, and Reactivation ...... 46
Figure 3-1. Self-Contained Breathing Apparatus from MSA ...... 50
Figure 3-2. Various Atmosphere-Supplied Respirators ...... 51
Figure 3-3. Cross-sectional View of Respirator Cartridge ...... 52
Figure 3-4. Different Designs of Air-Purifying Respirators ...... 55
Figure 3-5. Full-Face Air Purifying Respirators: Industrial and Military ...... 56
Figure 3-6. M50 JSGPM and Effect of Cartridge on Aiming of Weapon ...... 57
Figure 3-7. Joint Service Lightweight Integrated Suit Technology (JSLIST) ...... 62
Figure 3-8. NFPA 1991 and NFPA 1994 Permeation Test Cell ...... 75
Figure 3-9. ASTM F 903 Penetration Test Cell and Test Apparatus ...... 76
Figure 4-1. MIST Chamber at NC State University ...... 79
Figure 4-2. Passive Adsorbent Dosimeter and Structure of Tenax® TA ...... 84
xv Figure 4-3. Passive Diffusion through a Permeable Membrane ...... 85
Figure 4-4. PAD Placement Diagram for ASTM F 2588 ...... 90
Figure 4-5. PAD Placement Diagram for TOP 10-2-022 ...... 91
Figure 4-6. Dose-Response Curve for Vomiting as a Result of Intravenous dose of VX ...... 93
Figure 5-1. Extraction Methodology of Tenax® from PAD ...... 102
Figure 5-2. Modified SPE Tube Used in Tenax® Extraction from PAD ...... 103
Figure 5-3. Vacuum Manifold Used to Extract Methyl Salicylate from Tenax® ...... 104
Figure 5-4. Chromatogram of Low-Level Methyl Salicylate Standard at 310 nm ...... 111
Figure 5-5. Chromatogram of High-Level Methyl Salicylate PAD Extract at 310 nm ...... 112
Figure 5-6. Low-Range Methyl Salicylate Calibration Curve on HPLC at 310 nm (Equivalent to 3- 3,000 ng/PAD with a 4-µL injection) ...... 113
Figure 5-7. High-Range Methyl Salicylate Calibration Curve on HPLC at 310 nm (Equivalent to 12,000-120,000 ng/PAD with a 0.5-µL injection) ...... 113
Figure 5-8. Average Volume of Acetonitrile Collected After Extraction for Each Manifold ...... 117
Figure 5-9. Extraction Efficiency of Common Solvents ...... 119
Figure 5-10. Extraction Efficiency across Range of Expected Methyl Salicylate Amounts ...... 120
Figure 6-1. SolidWorks® Visualization of Bench-Scale Chamber Design...... 123
Figure 6-2. Bench-Scale MIST Chamber ...... 124
Figure 6-3. Chamber End Cap with Quick-Access Door Closed (left) and Open (right)...... 125
Figure 6-4. Methyl Salicylate Vapor Generation System for Bench-Scale MIST Chamber ...... 126
Figure 6-5. Flow Schematic for Bench-Scale MIST Chamber ...... 127
Figure 6-6. CIC Photonics Gas Cell FT-IR Measurement System ...... 128
Figure 6-7. Air Sampling System for Bench-Scale MIST Chamber ...... 128
xvi Figure 6-8. Bench-Scale MIST Chamber Concentration Profiles ...... 132
Figure 6-9. Relationship between MeS Boiler Flow Rate and Chamber Concentration ...... 133
Figure 6-10. Methyl Salicylate Concentration Profiles for Bench-Scale and Full-Scale Chambers .. 136
Figure 7-1. Passive Adsorbent Dosimeter Used in MIST Protocol. Dimensions of Active Sampling Surface Area are 2.5 cm x 1.8 cm According to ASTM F 2588...... 142
Figure 7-2. Calculated Percent Deviation from the Standard Length of 2.5 cm for 20 PADs ...... 152
Figure 7-3. Calculated Percent Deviation from the Standard Width of 1.8 cm for 20 PADs ...... 152
Figure 7-4. Variability in the PAD Surface Area between Lots (Error Bars Indicate the 95% Confidence Interval and Dotted Lines Indicate the Surface Area Tolerance According to ASTM F 2588) ...... 153
Figure 7-5. Total Methyl Salicylate Uptake (ng) Versus Exposure Ct (mg.min/m3). Each Data Point is the Average of Four PADs. Slope Corresponds to a PAD Uptake Rate of 14.3 cm3/min and a Film Uptake Rate of 3.4 cm/min ...... 154
Figure 7-6. Average Uptake Rate (cm/min) Versus the Time of Exposure (Minutes). Average Steady State Uptake Rate Approximately 3.5 cm/min...... 156
Figure 7-7. Calculated Uptake Rate (cm/min) Versus the Post-Exposure Storage Time for PADs With and Without Adhesive. All PADs Were Exposed for 30 minutes at 100 mg/m3 and Stored at 4°C...... 160
Figure 7-8. Total Amount of MeS (ng) Adsorbed for PADs Exposed for 30 minutes at 100 mg/m3. PADs Were Extracted Immediately, Stored Below -70°C in Dry Ice for One Day, and Stored at 4°C in a Refrigerator for One Day ...... 162
Figure 8-1. Bench-Scale MIST Chamber ...... 168
Figure 8-2. Fabrics and Sample Holders for Bench-Scale Exposures ...... 170
Figure 8-3. Methyl Salicylate Concentration Profiles for Bench-Scale Exposures ...... 175
Figure 8-4. Methyl Salicylate Uptake Rates for PADs and Fabrics ...... 176
Figure 8-5. Methyl Salicylate Adsorption of Spandex-Blended Fabrics ...... 178
xvii Figure 8-6. Measured Uptake Rates of Various Fabrics After 30 minutes of Exposure to 100 mg/m3 ...... 179
Figure 9-1. NEWTON Sweating Thermal Manikin (Measurement Technology Northwest) at North Carolina State University ...... 183
Figure 9-2. MIST Results from RMC and TNO Human-Manikin Comparison ...... 185
Figure 9-3. PTFE Sealant on the Manikin Head Post...... 187
Figure 9-4. MIST Manikin on Elliptical Walker in NCSU’s MIST Facility ...... 188
Figure 9-5. Physical Attachment Points Between Manikin and Elliptical ...... 189
Figure 9-6. Human Subjects in MIST Trial for Blauer® MIRT Class 3 Ensemble ...... 191
Figure 9-7. Geometric Mean PPDF Values for 8-Subject MIST Trials on the Blauer® MIRT Ensemble (Average of Three Replicates for Each Subject)(Dotted Line Indicates the NFPA 1994 Passing Local Protection Factor (120) for a Class 3 Ensemble) ...... 195
Figure 9-8. Comparison of Geometric Mean Systemic PPDF Values for Each MIST Trial with the Blauer® MIRT Ensemble (Error Bars Indicate the 95% Confidence Interval)(Dotted Line Indicates the NFPA 1994 Passing Systemic PPDF (76) for a Class 3 Ensemble) ...... 197
Figure 9-9. Effect of Number of Subjects on the Systemic PPDF Values for the Blauer® MIRT Ensemble (Error Bars Indicate the 95% Confidence Interval) ...... 198
Figure 9-10. Comparison of Geometric Means of PPDF Values for 6-Subject MIST Trials on Blauer® MIRT Class 3 Ensemble and LION® ICG Class 2 Ensemble ...... 200
Figure 9-11. Comparison of Geometric Mean PPDF Values for an 8-Human Subject and a 3-Manikin Subject MIST Trial on the Blauer® MIRT Class 3 Ensemble (Error Bars Indicate the 95% Confidence Intervals) ...... 202
Figure 9-12. Effect of Number of Human Test Subjects on the Variability in Systemic PPDF Values Compared to the Geometric Mean of Three Manikin Tests on the Blauer® MIRT Ensemble (Error Bars Indicate the 95% Confidence Interval) ...... 203
Figure 9-13. Geometric Mean Systemic PPDF Values for Three Replicates of Manikin and Human Subject MIST Trials on Blauer® MIRT Class 3 Ensemble (Error Bars Indicate 95% Confidence Interval) ...... 204
xviii Figure A-1. Calculation of Localized PPDF Values - Screen Shot from Body Region Hazard Analysis Program Developed at NCSU ...... 224
Figure A-2. Calculation of Systemic PPDF Values - Screen Shot from Body Region Hazard Analysis Program Developed at NCSU ...... 229
xix LIST OF EQUATIONS
Equation 4-1 Calculation of Uptake Rate Using Fick’s Laws of Diffusion ...... 86
Equation 4-2 Calculation of Median Effective Dose for HD Vesication for Body Region ...... 95
Equation 5-1 Low-Range Methyl Salicylate Calibration Curve for HPLC Analysis ...... 114
Equation 5-2 High-Range Methyl Salicylate Calibration Curve for HPLC Analysis ...... 114
Equation 6-1 Relationship Used to Calculate Methyl Salicylate Boiler Concentration ...... 133
Equation 7-1 ASTM F2588 Uptake Rate Calculation ...... 145
Equation 7-2 ASTM F2588 Calculation for Raw Protection Factor ...... 145
Equation A-1 ASTM F2588 PAD Uptake Rate Calculation ...... 223
Equation A-2 ASTM F2588 Film Uptake Rate Calculation ...... 223
Equation A-3 Calculation for Expsoure Dosage ...... 223
Equation A-4 ASTM F2588 Calculation for Raw Protection Factor ...... 225
Equation A-5 ASTM F2588 Calculation for Localized Physiological Protective Dosage Factor .. 225
Equation A-6 ASTM F 2588 Systemic Physiological Protective Dosage Factor Calculation ...... 228
Equation A-7 Military TOP 10-2-022 Mass Normalization Equation ...... 230
Equation A-8 Military TOP 10-2-022 Calculation for Raw Protection Factor ...... 232
Equation A-9 Military TOP 10-2-022 Local Agent Ct50 Calculation ...... 234
Equation A-10 Military TOP 10-2-022 Calculation for Clothed Effective Ct ...... 234
xx LIST OF ABBREVIATIONS
8I3M 8th International Meeting on Manikins and Modeling
AATCC American Association of Textile Chemists and Colorists
AChE Acetylcholinesterase
ACh Acetylcholine
ASTM American Society for Testing and Materials
BASF Badische Anilin- und & Soda-Fabrik
BRHA Body Region Hazard Analysis
CBDCOM U.S. Army Chemical and Biological Defense Command
CBRN Chemical, Biological, Radioactive, and Nuclear
Ct Exposure Dosage (Product of Concentration and Exposure Time)
CNS Central Nervous System
CWA Chemical Warfare Agents
DMMP Dimethyl methyl phosphonate
DMS Dimethyl sulfate
ED50 Effective Dosage for 50% of population
EPA Environmental Protection Agency
GA Tabun
GB Sarin
GD Soman
GF Cyclosarin
H Sulfur Mustard (30% Impurities) (Levinstein Process)
HD Distilled Sulfur Mustard (Mustard Gas)
xxi HN1 Nitrogen Mustard-1 [ethyl-bis(2-chloroethyl)amine]
HN2 Nitrogen Mustard-2 [methyl-bis(2-chloroethyl)amine]
HN3 Nitrogen Mustard-3 [tris(2-chloroethyl)amine]
ICI Imperial Chemical Industry
IDLH Immediately Dangerous to Life or Health
IPA Isopropanol
JSGPM Joint Service General Purpose Mask
JSLIST Joint Service Lightweight Integrated Suit Technology
LOD Limit of Detection
LOQ Limit of Quantitation
MDL Method Detection Limit
MeOH Methanol
MEDsys Minimum Exposure Dosage (systemic)
MEDHD Minimum Exposure Dosage (sulfur mustard HD)
MeS Methyl salicylate
MIST Man-In-Simulant-Test
NFPA National Fire Protection Association
OSHA Occupational Safety and Health Administration
PAD(s) Passive Adsorbent Dosimeters
PAM Pyridine aldoximine methiodide
PAPR Positive Air-Purifying Respirator
PNS Peripheral Nervous System
PPDF Physiological Protective Dosage Factor
xxii RMC Royal Military College
SCBA Self-Contained Breathing Apparatus
TIC Toxic Industrial Chemical
TNO The Netherlands Organization for Applied Scientific Research
TOP Test Operations Procedure
TNT Trinitrotolune
TPACC Textile Protection and Comfort Center
VX VX (V-Series Nerve Agent)
xxiii CHAPTER 1: Introduction and Purpose of Research
1.1 Purpose of Research
The Man-In-Simulant-Test (MIST) is intended to evaluate the protective performance of an entire chemical protective ensemble including the garment, respirator, gloves, boots, and any other piece equipment intended to protect the individual. Through the use of human test subjects, the ensemble is worn and stressed as it would be in the field during normal use. Although MIST methods have been around for almost 20 years, there has not been a large amount of research conducted on the test methods with results that have been published in open literature. Many of the MIST facilities are located at military institutions which makes it very difficult for industry, academia, and standards committees to further develop the methods. In July of 2008, the construction of a new MIST Facility at North Carolina State University was completed which made it the only facility of its kind at an academic institution in the United States. This new MIST facility is housed at the Center for
Research on Textile Protection and Comfort (TPACC). While this new facility provides an alternative testing location for both industry and military development, one of its main advantages is its location at a top tier research university. Since the underlying principle of this test method is to assess the protective performance of an ensemble to the most accurate degree possible, it is imperative that the research at TPACC’s MIST facility be focused on advancing the current methodologies and ensuring that the methods provide the most realistic and accurate evaluation of the ensembles that protect soldiers and first responders every day. With this principle in mind, the scope of this research is focused around not only questioning but improving the current methodologies as well as investigating potential advancements in the test itself.
1 1.2 Research Objectives
1.2.1 Advancement of the MIST Methodology
With very little research published in the MIST field, it is difficult to gain a full appreciation
of why the test was originally developed and how it was intended to be conducted. The first objective
of this research was to develop a comprehensive understanding of the history behind chemical
warfare, chemical protective clothing, applicable standards, and the MIST methodology. A point of
emphasis has been placed on investigating every aspect of the current methods to identify
improvements in the current procedures. The critical areas of interest were the development of a standardized extraction and analysis technique for methyl salicylate (simulant for mustard gas), characterizing the properties and handling procedures of the Passive Adsorbent Dosimeters (PADs) used to collect the simulant vapors inside the ensemble, and developing a bench-scale exposure chamber to conduct testing.
1.2.2 Development of Next Generation Manikin for Chemical and Biological Protection Research
The second phase of the research was focused on the development and incorporation of articulated thermal manikins into the MIST methodology. Standard MIST protocols require the use of multiple human subjects which introduce variability due to physiological differences between subjects. Thermal manikins offer controlled subject size, shape, surface temperature, sweating rate, and simulated work rate which can potentially provide a lower degree of variability. The main objectives for this phase of the research are to investigate the variability in human MIST results using two different ensembles of varying protection levels, to conduct MIST trials with the thermal manikin in one of the ensembles, and to develop a comparison between the protection results from humans and
2 manikins. The incorporation of the manikin system into the MIST protocol has a sub-set of objectives including an investigation into the methyl salicylate uptake rates of fabrics that may serve as a skin simulant and the development of a more realistic range of motion for the manikin. The overall goal for this phase of the research was to form a more accurate picture of how thermal manikins can feasibly be used in MIST testing. The critical aspect of this objective is the comparison between the manikin and human MIST evaluations.
3 CHAPTER 2: Chemical Warfare: History, Agents, and Toxicology
2.1 Introduction to the Modern Age of Chemical Warfare
2.1.1 Chemical Warfare in the Ancient World
A common misconception about chemical warfare is that it is a tactic that was recently
developed during the great world wars in the not so distant past. If asked to describe chemical
warfare most people in today’s society would mention words such as nerve agents, mustard gas,
weapons of mass destruction, and other terms that may seem sophisticated and esoteric to many
people. In reality, chemical warfare has been in existence since the first humans covered arrows and
spear tips with the natural poisons that they found around them to increase the effectiveness of
hunting. Recognizing the power that chemicals could have on living creatures, ancient man quickly
converted his hunting techniques to methods of protection, defense, and eventually to tactics of
warfare. There are many examples in the ancient world where people used everyday items and
common chemicals to gain an advantage on the battlefield. Many of the first warfare agents came
from poisonous plants or animals. In ancient Greece (590 BC) the Kleisthenes poisoned the water source of the city of Kirrha with black hellebore (Christmas rose), and due to the sickness that was brought on by drinking the tainted water, the people of the city were far too weak to fight back [1].
Poisons were not the only chemical agents used in the ancient world. In 660 BC, Greek fire was invented by Callinicus of Heliopolos. This mixture of naphtha, sulfur, petroleum, phosphorus, and other chemicals formed a thick tar that after set ablaze was nearly impossible to put out. This Greek fire would burn on the surface of water and cause massive damage to any ships or other vessels caught in its path [2]. These tactics were not isolated to the Mediterranean regions; indigenous
peoples of every part of the world had their own form of chemical warfare. The South American
4 Indians killed many Spanish conquistadors with their poison darts coated with lethal lipophilic
alkaloid toxins from frogs, and they also were reported to paint their fingernails with strychnine or
curare in the event they were forced into hand-to-hand combat [1].
2.1.2 Dr. Fritz Haber: Feeder of Nations and Father of Chemical Warfare
Although there were many cases of chemical warfare agents (CWAs) in the ancient world, the modern age that is most relevant to the current understanding of chemical warfare began in the early 1900s by a German scientist. As described in his biography, Dr. Fritz Haber was known as an extremely intense individual, who was very focused on being accepted into the German academic and military societies, even to the point of denouncing his Jewish heritage [3]. Society shuns Haber because he is known as the Father of Chemical Warfare, but it is important to realize the other impacts that he had on the entire world. Aside from being the mastermind behind the first chemicals used in modern wars, Haber was instrumental in allowing the growing world to feed all of its peoples.
In the late 1890s and early 1900s, Germany, along with most of the civilized world, was dependent on a strip of Chilean coastline two hundred and twenty miles long by five miles wide as its sole source of nitrates for fertilizer [4]. The nitrate rich guano deposits from bird droppings served as the world’s fertilizer for many years and seemed to be limitless to many people. As the world population began to rapidly increase, many scientists became worried about exhausting the current supply of fertilizer.
Without a means to enrich the soil, fields would not produce enough food to feed the people. It had been known for some time that diatomic nitrogen comprised a majority of the air that was all around.
It was Fritz Haber that invented a way to capture that nitrogen from the air, thus going down in history “as the man who by this means won bread from air and achieved a triumph in the service of his nation and all of humanity [3].” Haber’s process to combine nitrogen and hydrogen gases and
5 form ammonia revolutionized the way the world provided food to its growing population. By
partnering with Carl Bosch of Badische Anilin- und & Soda-Fabrik (BASF), one of Germany’s most successful chemical companies, the Haber-Bosch process was turned from a bench-top experiment into a full scale industrial process.
Although Haber was honored with the Noble Prize in Chemistry in 1918 for his work on nitrogen fixation from air [5], many people in the scientific community chose not to attend his award
ceremony in protest to his contributions to chemical warfare [3]. However, introducing chemical
agents into the battlefield was not the only effect that Haber had on the First World War. As with
many scientific breakthroughs, the Haber-Bosch process had cascading effects in other areas of the scientific and industrial worlds. Having a constant supply of ammonia within the borders of Germany allowed the country to produce gun powder and explosives without the fear of an Allied blockade of the Chilean nitrate source. This ability to produce munitions from its own resources allowed
Germany to extend World War I for at least one to two years [4]. For as much as Haber appeared to be an evil and diabolical mastermind due to his involvement in chemical warfare, he was more so an opportunistic scientist taking advantage of the byproducts of a flourishing industry at the time.
2.1.3 The Relationship between the Textile Industry and Chemical Warfare
The growth in the textile industry during the 1800s led to many advances in chemical processes and also spurred a significant growth in the chemical industry. Many procedures used in fabric preparation, dyeing, and finishing require chemicals such as caustic soda, soda ash, sodium hypochlorite, and dye compounds. By the 1900s, Germany had become the premier manufacturer of dyestuffs in the world, and most other countries were satisfied with importing dyes from German manufacturing plants. Around this same time, it was found that toluene-based explosives were more
6 effective as military weapons than the previously used nitroglycerine-based blasting powders that
were developed through Nobel’s patents [3]. Since the chemical reactions required to make
trinitrotoluene (TNT) are very similar to those involved in dyestuff manufacturing, Germany had a
significant advantage on the rest of the world. The machinery and processes in the German dyestuff
plants were easily converted over to those required for TNT production. The high toluene demand for
TNT production could not be met, but it was discovered that ammonium nitrate mixed with very little
TNT still provided an explosion just as violent as the pure compound with a fraction of the required
toluene. Again, Germany had the advantage with the already industrialized Haber-Bosch process pumping out massive quantities of ammonium nitrate used for fertilizers.
The dyestuff portion of the textile industry was not the only area that contributed to weapons of warfare. The processes that were used to produce soda ash and caustic had many byproducts that had no real purpose during that time. Hydrochloric acid gas, hydrogen, and chorine were all produced in vast quantities and were simply released into the atmosphere by many of the facilities.
This release of reactive chemicals into the atmosphere caused acid rain to destroy vegetation around the manufacturing plants. Some of the plants captured the chlorine and converted it with the aid of lime into a bleaching agent, but the vast majority was pressurized to its liquid state in cylinders so that it could be stored [3]. Since organic chemistry had not yet developed into a worldwide industry it did
not produce a high demand for chlorine, and the manufacturers found themselves with stockpiles of
the chemical. What was waste to the textile industry became the first modern chemical warfare agent
in the eyes of an opportunistic Fritz Haber.
7 2.2 The First Generation of Chemical Warfare Agent: Irritants, Chlorine, and Phosgene
2.2.1 The First Large Scale Use of Chlorine as a Chemical Warfare Agent
By the early part of 1915, both the Allied and German armies had essentially reached a
stalemate on the battlefield because all of the forces were held up in trenches. Neither side made any
major advances into the other side’s territory due to the nature of trench warfare tactics; mainly unloading massive amounts of gunfire and mortars into the opposition’s trenches until they were defeated or until one side had exhausted all of their munitions and resources. Chemical weapons were thought to be a potential solution to the stagnant war because they could force the soldiers out of the trenches and onto the open battlefield. Initially, artillery shells were filled with non-lethal irritant gases such as lachrymators and sternutators (tear gases and sneezing gases respectively). The French and German militaries were the first to use these gases in battle even though there had been prior agreements between major countries at the 1899 Hague Peace Conference prohibiting the use of unconventional weapons [6]. The non-lethality of the irritant gases served as the justification for their use. Once the French armies struck first with shrapnel shells filled with ethyl bromoacetate, the door was open and the German forces started to retaliate with irritants of their own [4]. Two of the
German tear gas artillery shells were referred to as T-Stoff, which contained xylyl- and benzyl bromide, and B-Stoff, a combination of T-Stoff and bromoacetone [6]. The French and Russian forces were attacked with these munitions, but they had very little effect. Generating a large cloud of the irritant that was concentrated enough to have any effect on the opposition was almost impossible using the crude artillery shells and firing tactics. This issue was one of the main reasons that chemical warfare was never a truly effective offensive weapon on the battlefield.
By December of 1914, the German authorities had appointed Haber as the chief of the chemistry section in the War Department for Raw Materials [4]. He took it upon himself to
8 determine the best way to incorporate chemicals into warfare to force the Allied troops out of the
trenches. According to Major S. J. M. Auld, the Chief Gas Officer in the British military and primary
educator to the troops at the time, an effective gas weapon should be 1) highly poisonous, 2) easily
and cheaply manufactured in large quantities, 3) be compressible to aid in transport, 4) be heavier
than air so it seeps down into the trenches, and 5) be stable enough so that it will only react with the
human targets that it should contact [7]. Thus, Haber turned to pure chlorine gas because it was very
toxic, it was in great supply in the German alkali plants, it could be transported in cylinders, and it could be released in an enormous cloud on the frontlines that slowly dispersed into the trenches. The only requirement that chlorine gas did not meet was the stability requirement which would ultimately be its downfall. After significant convincing, the military officials allowed Haber to put his plan into action, and he was in charge of training the gas-troops and determining where and when the attack would take place. Being a cloud of gas, the trajectory of the released chlorine was entirely dependent on the direction of the wind. Finally after many postponements due to the prevailing eastward winds over the battlefield in Ypres, Belgium, on April 22, 1915 Haber gave the order to release the chlorine gas from over 5,500 cylinders across a six kilometer stretch of land [4]. The estimated amount of four hundred tons of chlorine gas produced a yellow-green cloud approximately fifty feet tall and four miles wide that slowly moved toward the junction between the British and French lines [3]. The most effective aspect of the first chlorine attacks was the element of surprise and the panic that it generated in the unprepared soldiers. Common battlefield practice was to head into the trenches when under attack. Since chlorine is more dense than air, the gas cloud fully enveloped these areas, and forced the soldiers out of the trenches where they were met with the German gunfire. The Allied forces were ill-equipped to deal with this novel weapon on the battlefield, and a large four to five mile wide opening was made in their front line. Fortunately for the Allied forces, the German military did not
9 follow the gas attack with sufficient infantry to make a permanent gain in territory. It is believed that the military officials were skeptical of the effectiveness of the gas attack and therefore did not plan to provide appropriate numbers of troops to seize the large opening in the French and British lines [4].
This skepticism led to somewhat of a failure for the first gas attack, and took away the main advantage that this new chemical warfare provided: the element of surprise.
2.2.2 Chlorine: Mechanism of Action
In most cases chlorine is found bonded to other elements such as in salts with sodium and calcium, or with hydrogen and oxygen in hydrochloric (HCl) or hypochlorous acids (HOCl). This high reactivity is the main property responsible for chlorine’s mechanism of action as a chemical agent. Like the previous chemical agents used in the T- and B-Stoff artillery shells, chlorine is also an irritant. When inhaled, chlorine reacts with the water that is held in the epithelial lining of the upper respiratory tract and forms irritating acids [8]. If the exposure dosage if significant, the individual can suffer respiratory failure from pulmonary edema. The irritation causes the lungs to fill with liquid which essentially drowns the individual. Many of the victims of chlorine attacks showed symptoms of oxygen starvation such as blueness of the face and lips [9].
2.2.3 Protection Against Chlorine: First Respirators Used in Warfare
Prior to the first chlorine gas attacks there were no significant respirators used in the military, and the only civilian respirators were mainly used for the mining industry. The earliest known design for a protective mask or respirator was describe by Leonardo da Vinci in one of this research notebooks [10]. Da Vinci’s protective mask was simply a cloth that was soaked in water and held to
10 the individual’s mouth. This very simple design was not far off from the very first respirators that
were used in response to the German chlorine attacks. After the gas was identified as chlorine, the
first response was to tell the soldiers to soak rags or cloth scraps in a sodium bicarbonate solution that
had been delivered to the trenches. This solution would provide some protection from the gas attacks
by neutralizing the chlorine [4]. Just days after the first attack, Winston Churchill suggested that cotton wool pads could be wetted and placed over the mouth, much like the design that the Navy used in smokescreen experiments. Even after being informed that moistened cotton wool was nearly impossible to breathe through, the War Office sent out a call through the newspaper requesting the women in Britain to provide cotton wool pads that could be used as respirators. These pads became known as The Daily Mail respirators, and just as people suggested, they were completely useless because the soldiers could not breathe through them [9]. The first major deployment of a protective mask that helped to save many lives was the distribution of the British Black Veiling Respirator
(Figure 2-1). Named after the material that it was constructed from, the respirator consisted of cotton waste packed into a pocket that was sewn into a black veiling material. The respirator was soaked in a solution of sodium thiosulfate, sodium bicarbonate, and glycerol. The material was placed over the nose and mouth and tied around the head of the individual, but it had significant problems [4]. The effectiveness of the neutralizing solution was quickly exhausted, and the simple design did not provide a very tight seal around the face. The respirator would work for possibly five minutes before it needed to be re-soaked in the ‘hypo’ solution, but the gas attacks would last 45 minutes or more [9].
11
Figure 2-1. British Black Veiling Respirator [11]
2.2.4 Development of More Effective Respirators and More Lethal Agents
World War I is referred to by many historians as the “Chemists’ War” because as soon as the chemical agents were introduced onto the battlefield, the struggle to provide effective chemical counter-measures began. Chemists from every country involved were put to work to determine the best methods to neutralize the ever increasing lethality of the opposition’s next chemical agent. This battle between inventing better protection and developing more effective and lethal weapons has always been a fundamental struggle in warfare. First the weapon was a spear and the protection was a shield, then guns and bullets were introduced and bullet-proof vests were invented. Chemical warfare spurred the development of the entire industry of personal protective equipment. As soon as the
12 British and French scientists introduced their first methods of respiratory protection, the Germans began to formulate more effective chemical weapons that would defeat the gas masks.
After the limited success of the Black Veiling Respirator, the military officials and scientists decided that an apparatus that covered the entire head of the soldier was needed. The ‘Hypo’ Helmet
(Figure 2-2) was developed by Captain Cluny MacPherson in June 1915, and it consisted of a wool flannel material or bag that was soaked in the same ‘hypo’ solution previously used [4] [9]. A small window made from mica or triacetyl cellulose, allowed the soldier to see when wearing the helmet.
Although the protection provided by the ‘Hypo’ Helmet was better than the Black Veiling Respirator, the sensory deprivation caused by cloudy window hindered the movement and effectiveness of the soldiers on the battlefield.
Figure 2-2. British ‘Hypo’ Helmet [11]
13 The Germans quickly discovered that the ‘Hypo’ Helmet was not air tight and that there was a small volume of air that remained inside the helmet. Their method to defeat the ‘Hypo’ Helmet was to include the tear gas agent, chloroformate, in their assaults. Due to the intense eye irritation and pain caused by the tear gas, the Allied soldiers ripped their respirators off in an attempt to achieve some form of relief. The British countered this attack by adapting the ‘Hypo’ Helmet to include tight fitting goggles that would protect the eyes.
Knowing that the British masks used the ‘hypo’ solution to neutralize the chlorine gas, the
German scientists experimented with agents that would not be affected by the chemicals. Phosgene
(carbonyl dichloride, COCl2) was the next gas that was chosen by the Germans to use against the
Allied forces. At this point in the war, the differences between the British and German chemical warfare policies became evident. Since the Germans believed that Britain did not have the industrial capacity to manufacture the newest warfare agents, they did not improve upon their own original respirators as they developed new agents. The British scientists, while developing new agents themselves, made it a point to incorporate new protective measures in the event that the Germans had developed the same weapons [4]. Both sides were experimenting with phosgene at the same time, but the British developed a series of P Helmets. As previously stated, phosgene was not neutralized by the ‘hypo’ solution that was used with the other respirators so a new doping solution was needed.
The P Helmets were named after the sodium phenate solution that was used to neutralize both phosgene and hydrogen cyanide. After a recommendation from Russian scientists, the PH Helmet
(Figure 2-3) was developed by including hexamethylene tetramine in the doping solution because it was more effective at neutralizing phosgene [9]. The neutralizing solution was not the only difference between the P Helmets and the ‘Hypo’ Helmets. The P Helmets were constructed of an alkali resistant cotton flannel because the wool flannel of the ‘Hypo’ Helmets was degraded by the
14 caustic phenate solution. The new P Helmet design incorporated two circular glass eyepieces and an exhalation tube. The carbon dioxide in the exhaled breath was found to negatively affect the neutralization properties of the phenate solution, so the tube and valve allowed for the solider to stay
in the P Helmet for a much longer period of time.
Figure 2-3. Machine gunners wearing the PH Helmet [12]
After the first use of phosgene, the German’s realized that the British had developed the PH
Helmets and that they were fairly successful at neutralizing their new chemical agent. However, it was soon apparent that the PH Helmets shared the same basic flaw as all of the previous respirators
15 that the British had developed. These gas masks could be overcome by prolonged bombardment with the chemical agent. All of the respirators that have been discussed utilized a doping solution that was applied to the mask and therefore they all had very little neutralizing capacity. The most significant advancement in the British respirator design was the development of the Large Box Respirator
(Figure 2-4) because it provided a much higher capacity for the chemical agents. The Large Box
Respirators, as their name implies, consisted of a box that was filled with solid sorbents such as charcoal that sequestered the chemical agent instead of attempting to neutralize it. Using solid materials in the box allowed for the incorporation of new sorbents much easier than with the liquid doping solutions. The British also included cotton wadding in the box respirators to serve as a particulate filter which coincidentally protected the Allied soldiers from the effects of the German sternutators.
Figure 2-4. Large Box Respirator (left) [13] and Small Box Respirator (right) [9]
16 These sneezing agents were solid particles so they were not adsorbed by the charcoal filters, and the
Germans had hoped including them in with the other chemical agent attacks would defeat the box
respirators [4]. The main problem with the Large Box Respirators was the fact that they were very
large and cumbersome. The solution to this problem was to make a more efficient and much smaller
version appropriately named the Small Box Respirator (Figure 2-4).
The German respirator, as previously mentioned, remained mostly the same due to the
German beliefs that they had superior industrial processes and that the British could not quickly
manufacture new chemical agents on a large scale. The basic German respirator design was called
the Gummimaske (Figure 2-5), which means rubber mask. Their respirator contained a filter cartridge
on the front of the mask packed with charcoal which made it fairly effective against most agents.
However, the heavy filter cartridge on the front of the mask pulled the entire face piece away from the
individual’s face and allowed for more leakage. Also the cartridge was very small and could not be
used for extended periods of time. The British were able to exploit this flaw by maintaining a
prolonged low rate of fire so that the German troops had to remain in their protective masks for very
long periods of time. One method of maintaining a highly toxic concentration of the chemical agent
for a long period of time was through the use of the Livens Projector (Figure 2-6) and Stokes mortar.
The British developed the Livens Projector to alleviate the problem that attacking with gas presented.
Since chlorine gas released from cylinders only followed where the wind took it, there was a
significant amount of uncertainty when planning an assault. The Livens Projector solved this problem because it allowed the British to load full cylinders of a chemical agent into a firing cell, and launch the entire cylinder into the German trenches.
17
Figure 2-5. German WWI Gas Mask (Gummimaske) [14]
Figure 2-6. British Livens Projector [9]
18 2.3 The Second Generation of Chemical Warfare Agents: Vesicants
2.3.1 Unveiling of Vesicant Agents to the Battlefield
By the early part of 1917, the Germans had realized that their phosgene attacks were not as
effective against the more superior British respirators. The development of a new class of agents that
would not be neutralized by the filters in the respirators was an area of particular interest to Fritz
Haber and the German War Office. On July 12, 1917 the Germans unveiled their newest chemical
agent which had a vastly different mechanism of action than the previously used irritant gases.
Where chlorine, phosgene, lachrymators, and sternutators all primarily attacked the respiratory tract
and eyes, the target organ for the vesicant agents was the skin. By definition a vesicant is an agent
that causes blistering at the area of contact. German scientists, Dr. Lommel and Prof. Steinkopf,
suggested the use of a vesicant which they called ‘LOST’ (the name being derived from the first two letters in their names). This new vesicant was also referred to by many names including: Yellow
Cross by the Germans, Yperite by the French, and the British forces knew it as mustard gas or sulfur mustard [9].
When deciding on what chemical should be the vesicant of choice, the Germans looked at more than just the toxicity of the agent. The main decision was between sulfur mustard and dimethyl sulfate (DMS) [4]. The blistering effect of DMS was slightly less than that of mustard gas, but DMS was a commonly used industrial chemical. The Germans had already used phosgene which was an easily attainable chemical, and the British were quick to secure their own stockpiles of the weapon.
The deciding factor for which agent the Germans chose was once again the textile industry. Mustard gas was fairly easy to manufacture on a large scale using processes that were very similar to those already used to produce the German dyestuffs. Since the Germans still held a monopoly on the dyestuff production in Europe, they could quickly convert their manufacturing plants over to produce
19 mustard gas while the Allied forces would not have the means to produce the agent in a large supply.
Even after the war was over, many British officials sought for the German dyestuff industry to be disbanded in the event that Germany attempted to manufacture the vesicant again. The potential production of this agent also caused the British government to invest greatly into developing their own dyestuff industry [4]. The textile factor in the war even caused Fritz Haber to tell the German generals that they should not use the new vesicant unless they could guarantee that the war would be over in a year [3]. His fear and later reality was that within this timeframe the British would be able to develop and manufacture the agent in large supply. Haber was not worried about the fact that the
British would retaliate with mustard gas. His main concern was that mustard gas is a very oily and persistent chemical and it soaks into clothing and boot leather. He knew that the British would be able to supply new uniforms when their troops’ existing equipment was contaminated, but isolated
Germany did not have the resources to replace the equipment fast enough. Even after Haber expressed his caution, in 1917 Germany unleashed an offensive that consisted of a ten-day attack using 1,000,000 artillery shells containing over 2,500 tons of mustard gas which within three weeks had caused more Allied casualties than all of the previous year’s chemical assaults [4].
2.3.2 Sulfur Mustard: Physical Properties and Effectiveness of Use
The most common vesicant that was used in World War I was bis(2-chloroethyl) sulfide known as sulfur mustard or mustard gas. Both monikers provide some level of misconception when referring to the agent. The ‘mustard’ term is applied to this vesicant only because it has a slight odor of horseradish or garlic as observed by some of the first soldiers that were exposed. Referring to the agent as a gas is another misnomer that can cause confusion. The vapor pressure and freezing temperature of sulfur mustard, as can be seen in Table 2-1, show that under normal conditions the
20 agent is found in the liquid state. The high freezing point of 46-53°F was a major problem for using
sulfur mustard as an offensive weapon during the war because in most cases it was not in the vapor
state. Other agents such as lewisite, which is discussed later in this section, were commonly mixed
with mustard gas in order to lower the freezing temperature to approximately 10°F so that the agent
could be used in colder climates or delivered by aerial attack [15]. The first versions of mustard gas that were introduced were only about 70% pure sulfur mustard and 30% impurities. For clarity purposes, this version of sulfur mustard is commonly referred to as H, and it was synthesized using the Levinstein process with sulfur monochloride as a starting point. The amount of impurities found
in this mustard was a great disadvantage to using the agent in ballistic shells because the impurities
would settle out into a dense sludge that would alter the trajectory and flight of the shell [4]. It was not until the Second World War that processes were developed to synthesize a more purified version of sulfur mustard which is known as HD or distilled mustard. The distilled mustard did not have the characteristic brown-yellow color or the distinctive odor which made it very difficult to identify or realize a potential exposure.
Just as with the previous gases that were used, mustard gas stays low to the ground due to the
5.4-fold greater density than the surrounding air [15]. The density coupled with the high freezing point allowed for mustard gas to persist in the attacked area for a long period of time. Most attacks occurred during the cooler evening and night hours so the vapor condensed and stuck to the ground.
The next morning soldiers removed their respirators believing that they were no longer in danger, but as the sun rose and warmed the area the mustard gas re-vaporized and caused many casualties [7].
21 Table 2-1. Physical Properties and Chemical Structures for Common Vesicants [16]
Vapor Pressure Liquid Density Freezing Agent Common Name Chemical Name Chemical Formula Color Chemical Structure at 25°C at 25°C Point
Mustard bis(2-chloroethyl) 3 8-12°C Amber to dark H C4H8Cl2S 0.08 mm Hg 1.27 g/cm (70%) sulfide (46-53°F) brown
Mustard bis(2-chloroethyl) 3 14°C Clear to pale HD C4H8Cl2S 0.11 mm Hg 1.27 g/cm (distilled 96%) sulfide (57°F) yellow
ethyl-bis(2- -34°C Clear to pale HN1 Nitrogen Mustard-1 (ClCH CH ) NC H 0.25 mm Hg 1.09 g/cm3 chloroethyl)amine 2 2 2 2 5 (-29.2°F) yellow
methyl-bis(2- 1.15 g/cm3 -60°C Pale amber to HN2 Nitrogen Mustard-2 (ClCH2CH2)2NCH3 0.43 mm Hg chloroethyl)amine (at 20°C) (-76°F) yellow
3 -3.7°C Clear to pale HN3 Nitrogen Mustard-3 tris(2-chloroethyl)amine N(ClCH2CH2)3 0.01 mm Hg 1.24 g/cm (25.3°F) yellow
dichloro (2-chloro-vinyl) 3 -18°C Amber to dark L Lewisite C4H2AsCl3 0.58 mm Hg 1.89 g/cm arsine (-0.4°F) brown to black
22 It is almost certain that Germany introduced mustard gas as an offensive weapon, but in reality the persistent nature of the agent made it better served as defensive weapon. If trenches or tracks of land were sprayed with the vesicant then that area was essentially referred to as “no-man’s land” because anyone that entered the area would be exposed to the agent [7]. In addition, just as
Haber suggested, mustard gas penetrated the uniform materials, the leather of the boots, and any other
clothing that the soldiers wore making it impossible to use that equipment without receiving a
chemical burn. The persistence of the chemical made it very difficult to determine if an area was still
contaminated, but the property of mustard gas that made it receive its insidious reputation was the
latent period between exposure and pain realization.
Exposure to the previous chemical weapons was very easy to recognize because pain
occurred very soon after, if not immediately following, contact with the agent. The instinctive
response to flee from the pain and protect oneself was not evident upon exposure to mustard gas.
Many accounts suggest that the soldiers may have caught a slight hint of the mustard odor, but
experiencing no immediate pain or threat they did not take any precautions to protect themselves.
The latent period between exposure to mustard and onset of symptoms can be anywhere from hours to days. The exposed soldiers would return to their bases and begin to have skin, lung, and/or eye irritation that progressively worsened until they could not continue their daily routines. This ability to cause massive casualties was the foremost reason that mustard gas was used so extensively during the
First World War. During its heyday, the main objection to chemical warfare was that it caused near instantaneous death on a grand and gruesome scale. This viewpoint was mostly propaganda that was driven by the media outlets during that time. In reality, chemical warfare’s main purpose was to produce casualties not fatalities. The basic reasoning behind this statement is that soldiers that died on the battlefield could in essence remain there and have no significant impact on the rest of the
23 battle. However, an injured solider must be carried out of battle, medically attended to, and ultimately tie up the enemies resources for days, weeks, or months on end [4]. Chemical warfare even caused the relocation of resources to newly formed decontamination or medical units and also caused psychological burdens and fatigue to set in with all of the soldiers due to the necessity of wearing cumbersome and restrictive protective clothing. In this understanding, mustard gas became the king of the war gases because it was capable of removing substantial numbers of active soldiers from the field, and it also provided a means to contaminate and deny an entire area for long periods of time. This fact is illustrated in Table 2-2 through the use of the casualty and fatality statistics attributed to chemical warfare. The initial introduction of chlorine and phosgene caused many casualties but also many fatalities. The emergence of mustard gas saw an order of magnitude increase in casualty numbers but still relatively the same number of fatalities. Only 2% of the soldiers that came into contact with mustard gas during the given time frame actually died from the exposure.
Table 2-2. Ratio of Chemical Warfare Fatalities to Chemical Warfare Casualties [4]
Period Prime Weapon Casualties Fatalities Ratio (%)
1915 First chlorine attacks 10,000 3,000 30.0
1915-1916 Chlorine in ascendance 4,207 1,013 24.1
1916-1917 Diphosgene rising 8,806 532 6.0
From July 1917 Diphosgene in ascendance 18,134 1,859 10.3
Mustard Gas 124,707 2,308 1.9
24 Many firsthand accounts of vesicant exposure were recorded and the following excerpt (taken from Chemical Warfare Agents: Toxicology and Treatment [17]) clearly shows the ability of mustard gas to cause severe damage to the exposed individual. The following is an account from Adolph
Hitler who was exposed to mustard gas in October 1918. It is believed that Hitler’s reluctance to employ chemical weapons during World War II could be tied back to the excruciating pain that he experienced during this attack.
During the night of October 13-14th (1918) the British opened an attack with gas
on the front south of Ypres. They used the yellow gas whose effect was unknown
to us, at least from personal experience. I was destined to experience it that very
night. On a hill south of Werwick, in the evening of 13 October, we were
subjected to several hours of heavy bombardment with gas bombs, which
continued through the night with more or less intensity. About midnight a
number of us were put out of action, some fore ever. Towards morning I also
began to feel pain. It increased with every quarter of an hour, and about seven
o’clock my eyes were scorching as I staggered back and delivered the last
dispatch I was destined to carry in this war. A few hours later my eyes were like
glowing coals, and all was darkness around me. [17]
2.3.3 Additional Vesicant Agents
Sulfur mustard was not the only vesicant agent that was developed during the war. As seen in Table 2-1, chemists were quick to modify the basic chemical structure of sulfur mustard by replacing the sulfur atom with a nitrogen atom thus producing the nitrogen mustards. Although these
25 agents do have similar vesicating properties to sulfur mustard, they were not used as chemical warfare
agents in any significant role [16]. The nitrogen mustards and their derivatives have actually found
use as chemotherapeutic agents and other pharmaceutical drugs.
Besides sulfur mustard, the only other well-known vesicant developed and manufactured by numerous countries was lewisite. This arsenic-based vesicating agent was developed during 1918 in the United States by a team of scientists at the Catholic University in Washington D.C. headed by Dr.
Winford Lewis [16]. Lewisite was the American response to chemical warfare and was touted as a much more potent vesicant than mustard gas. Although lewisite did cause effects to appear much more quickly than mustard gas, the resulting blisters were usually less severe and they healed much quicker. Other downsides of the agent were that it did not penetrate materials as effectively or persist in the contaminated area for long periods after exposed. Even with the known disadvantages many countries produced stockpiles of the so-called “more potent” vesicant throughout both world wars, but there are very few accounts of lewisite ever being used on the battlefield [4].
2.3.4 Basics Principles of Percutaneous Absorption
To appreciate how vesicants cause blistering to occur on the skin of an exposed individual, it is first imperative to have a basic understanding of skin anatomy and dermal absorption. Human skin has a very complex composition and makes up the largest organ in the human body. On an average human adult the skin can have a surface area of 1.5 to 2.0 m2 which is a substantial area for exposure to and penetration of chemicals [18]. The distribution of surface area per body region is given in
Table 2-3 for an adult and an infant. In both cases, the legs and trunk make up the majority of the
surface area of skin and therefore have a higher probability of being exposed to the environment. The
different body regions have varying degrees of susceptibility to potential threats depending on many
26 different characteristics of the skin that covers the area. In general, the rate of toxicant penetration in the varying body regions is as follows [19]:
Scrotal > Forehead > Axilla > = Scalp > Back = Abdomen > Palm and Plantar
Therefore, the genital or scrotal regions, although comprising only 1% of the total body surface area, are of significant interest for protection and risk assessment because they have the highest penetration rates of all the body regions. Alternatively, the hands and feet have the lowest penetration rates and therefore provide a higher natural protection than other regions of the body. The differences in rates of penetration across the body are mostly attributed to the composition of the skin that is in each area.
Table 2-3. Percentage of Body Surface Area for Anatomical Regions [18]
Anatomical Adult Neonate Region Body Area (%) Area (cm2) Body Area (%) Area (cm2) Genitals 1 190 1 19 Arms 18 3,420 19 365 Legs 36 6,840 30 576 Trunk 36 6,840 31 595 Head 9 1,710 19 365
Totals 100 19,000 100 1,920
As seen in Figure 2-7, the skin has multiple layers, components, and potential pathways for absorption. The structure of mammalian skin can be divided into three basic layers: the epidermis, the dermis, and the hypodermis or subcutaneous fat layer. Skin thickness can vary widely among different species, races, genders, individuals, and within anatomical locations on a single person. The
27 thickness of an average young adult’s skin can range from 0.5 mm in the eyelid to 10 mm on the sole
of the foot [20]. The variation in skin across the body also extends into each of the individual layers.
The epidermis which can range in thickness from less than 0.1 mm to 0.8 mm is further divided into
the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale.
Figure 2-7. Anatomy of Human Skin [21]
For the purposes of protection against the environment, the stratum corneum is the most important layer of the epidermis because it provides approximately 80% of the physical and chemical barrier properties of the skin [19]. The dermis, which is comprised of a thin papillary layer and a thicker reticular layer, can vary in thickness between 0.6 mm and 3.0 mm [22]. The subcutaneous fat layer
28 which is mainly comprised of the vasculature, fat, and connective tissues can be as thick as 3.0 cm in
certain areas of the body [22]. Many studies have been conducted to measure the variability of both
the epidermal and dermal thicknesses across different body regions. Lee et al. recorded
measurements across genders and body regions for Korean adults and compared them to previously
gathered data on Caucasian subjects [23]. These comparisons, given in Table 2-4, show how the
epidermis in Caucasians makes up approximately 4% of the total skin thickness, but it accounts for
roughly 8% in the Asian subjects. On a more focused scale, Hummel et al. reported the measured
thicknesses of the individual skin layers that are found in various parts of the hand. Even in this small
area of the body, the variation in the thickness of the epidermis from the back of the hand to the palm
side is significant (85 µm for the back of the hand and 550 µm for the palm) [24].
As previously stated, the stratum corneum of the epidermis provides the majority of the skin’s
resistance to penetration and absorption, and the differences in epidermal thickness account for a large percentage of the body region variation. The dermis and subcutaneous fat layer do not contribute significantly to the protective ability of the skin. Since these layers contain blood vessels they provide a means of systemic distribution, and if the toxicant penetrates the epidermis it is most likely going to be circulated throughout the body. The rate of toxicant penetration is not only affected by the thickness of the epidermis; it is also affected by the presence of appendages such as hair follicles and sebaceous glands. There are four basic routes of absorption through the skin: intercellular, transcellular, transfollicular, and sweat pore routes [19]. The intercellular route is accepted as the primary mechanism of dermal absorption with the other three routes only serving minor roles. The intercellular route of absorption is most often described by the brick-and-mortar model (Figure 2-8), where the “bricks” represent the protein rich corneocytes and the “mortar” refers to the intercellular lipids that make up the stratum corneum [19].
29 Table 2-4. Variation in Epidermal and Dermal Thickness between Races [23], [25], [26]
Epidermis (µm) Dermis (µm) Epidermis Percentage of Total Thickness Body Region Caucasian Korean Caucasian Korean Caucasian Korean Southwood et al. Artz et al. Lee et al. Southwood et al. Artz et al. Lee et al. Southwood et al. Artz et al. Lee et al. Chest 44 39 98 1,400 1,319 1,337 3.1 2.9 6.8 Back 66 62 76 1,805 1,911 1,941 3.5 3.1 3.7 Abdomen 41 40 79 1,640 1,492 1,248 2.4 2.6 6.0 Medial arm 42 44 69 956 1,027 943 4.2 4.1 6.8 Lateral arm 50 49 83 1,346 1,134 1,030 3.6 4.1 7.5 Medial forearm 52 48 74 994 995 1,123 5.0 4.6 6.2 Lateral forearm 55 53 102 1,060 953 1,077 4.9 5.3 8.7 Medial thigh 54 47 87 1,104 1,059 1,058 4.7 4.3 8.2 Lateral thigh 57 60 94 1,357 1,150 1,217 4.0 5.0 7.2 Posterior thigh 57 64 102 1,156 1,176 1,006 4.7 5.2 9.2 Medial leg 55 50 91 1,030 1,119 921 5.1 4.3 9.0 Lateral leg 58 60 109 1,077 1,009 1,013 5.1 5.6 9.7 Posterior leg 58 63 129 1,183 1,264 981 4.7 4.8 11.6 Mean 53 52 92 1,148 1,201 1,146 4.2 4.3 8.3
30 Toxicants traverse the pathway between the corneocytes following Fick’s laws of diffusion but the actual distance traveled can be ten times that of the epidermal thickness [19]. This extended pathway provides another means of protection for the underlying layers of skin. The “dead cells” or corneocytes that make up the stratum corneum are more lipophilic relative to the mainly aqueous layers of the epidermis and dermis. For this reason, highly lipophilic chemicals can remain in the stratum corneum and slowly traverse the absorption pathway. The transcellular route is considered minor because it refers to diffusion directly through cells which is a difficult process that is greatly affected by the polarity of the chemical. Hair follicles and sweat pores are also considered minor routes because the majority of the exposed epidermis does not contain these appendages [19].
Figure 2-8. Brick-and-Mortar Model of Dermal Absorption [19]
31 2.3.5 Vesicants: Mechanism of Action and Toxicity
Sulfur mustard can be characterized as an alkylating agent on account of its ability to transfer an alkyl or hydrocarbon group from one molecule to another. The highly reactive nature of sulfur mustard is due to the molecule’s ability to internally cyclize an ethylene group to form a strained three-membered ring known as an episulfonium ion. This highly reactive species is an electrophile that can go on to react with any number of nucleophiles in the many peptides, proteins, RNA, DNA, and other macromolecules that are found in the body [15]. Most of the vesicating agents contain bi- or tri-functionalities, meaning that one molecule can form two or three of the reactive species and effectively form cross links between DNA strands or other macromolecules [17]. The capacity to alkylate macromolecules is the main property allowing the nitrogen mustards to be used as chemotherapeutic agents in cancer therapy.
The actual mechanism of action from vesicant exposure to blister formation is a complex process that is still being researched [15]. In a very basic explanation, when skin is exposed to sulfur mustard or other vesicants, the junction between the epidermis and dermis is disrupted resulting in the formation of blisters. Since sulfur mustard forms an extremely reactive species that can chemically alter numerous sites on and within the cell, it is of no surprise that the final response is a summation of many different processes occurring simultaneously. Hurst et al. list some of the most investigated pathways which include: thiol depletion resulting in intracellular calcium imbalance, alkylation of
DNA, lipid peroxidation, and induction of the inflammatory response [15].
Any route of exposure to a vesicant, whether it is to the eyes, through the respiratory tract, or through dermal absorption can lead to the same alkylation-based consequences in the body. Exposure to the skin causes blisters to form on a local basis or where ever the vesicant made contact, but there are also systemic effects that have been observed long after exposure. The effect of skin variability in
32 different body regions can be seen in the distribution of documented injuries from World War I
(Table 2-5). Gilchrist et al. documented the mustard gas related injuries for 6,980 casualties, and it
can be seen that over 75% of the casualties received injury to the eyes and respiratory tract [27].
These data support the notions that mustard gas exposure was difficult to recognize because it appears that almost all of the affected soldiers were not wearing respirators to protect their eyes and respiratory tract. The latent period after exposure most likely exacerbated the injury to the eyes and lungs because the soldiers were not alarmed and therefore did not don their protective equipment.
Table 2-5. Distribution of Mustard Gas Injuries on Bodies of World War I Casualties [27]
Body Part Reported Injuries (%) Eyes 86.1 Respiratory Tract 75.3 Scrotum 42.1 Face 26.6 Anus 23.9 Back 12.9 Armpits 12.5 Neck 12.0 Arms 11.7 Chest 11.5 Legs 11.4 Buttocks 9.8 Abdomen 6.4 Thighs 6.0 Hands 4.3 Feet 1.5
33 Another reason that could explain the higher incidence of eye and respiratory injury is that these body regions do not have the luxury of being covered by the protective skin. In general, the
penetration of a toxicant can be related to the thickness or distance that must be traveled to be
absorbed into the body. The multiple layers of skin provide a large distance that the toxicant must
travel before it is picked up by the blood stream. On the other hand, the main function of the lungs is
to exchange oxygen and carbon dioxide in the blood stream, and to facilitate this process the distance
between the alveoli in the lungs and the blood in the capillaries is only a few cells wide. Therefore,
the process of systemic distribution of a toxicant occurs much more rapidly in the lungs. The eyes are
equally as sensitive because if the eyes are open, the skin of eyelids can do very little to restrict
exposure to a toxicant.
Assuming that an exposed soldier was wearing adequate respiratory protection, the next most
frequent locations of injury were the around the groin and face. As mentioned previously, these areas
are more susceptible to toxicant penetration, and they generally have thinner skin than other body
regions. While the thickness of the skin greatly affects the rate of penetration, other environmental
factors such as temperature and moisture also play a major role in the development of an injury.
Warm, moist areas of the body such as the groin are highly susceptible and can be greatly affected if
exposed to mustard gas. Also the need for elimination and defecation increase the likelihood that an
exposed individual will receive chemical burns in this area [28]. This particular susceptibility to mustard gas exposure was one of the reasons that the British military finally banned the wearing of kilts on the battlefield in 1940 [29].
Normally, exposure guidelines to a chemical or toxicant are givens in terms of mg/kg of body
weight or concentration units. However, when referring to chemical agents that are hazardous in the vapor state it is more appropriate to use the exposure dosage value or Ct. The Ct is a product of the
34 concentration of the toxicant and the time the individual is exposed expressed in units mg.min/m3. In
this manner, a short-term exposure to a high concentration of mustard gas would yield equivalent damage to the individual as a long-term exposure to a low concentration of the vesicant. In other words, a Ct of 1,000 mg.min/m3 could be achieved from either a 10-minute exposure to 100 mg/m3 or
a 100-minute exposure to 10 mg/m3. As the exposure dosage increases, the individual will experience
side-effects or injuries that also increase in severity. Depending on the route of exposure, the same Ct
value can cause varying degrees of damage with the eyes being the most sensitive, followed by the
respiratory tract, and finally the skin requiring much higher exposures for incapacitation or death. A
summary of the dose-response profiles for each route of exposure to sulfur mustard is given in Table
2-6. The symptoms associated with different threshold levels, the latency period, and significance of injury are given for the skin (Table 2-7), eyes (Table 2-8), and respiratory tract (Table 2-9).
Table 2-6. Dose-Response Profiles by Route of Exposure to Sulfur Mustard at 16-27°C [30]
Eye Respiratory Tract Skin Vapor Vapor Vapor Response Liquid (mg.min/m3) (mg.min/m3) (mg.min/m3) Threshold: First indications of non- 10-20 µg/cm2 ≤ 12 12-70 50 disabling signs and 35 mg/man symptoms
No estimates Injured but not disabled 50-100 < 100 100-300 reported
Incapacitation:
Inability to perform ICt50 = 200 ICt50 = 200 ICt50 = 1,000-2,000 770 mg/man designated duties
3,000-7,000 Death or permanent injury > 800 LCt = 1,000-1,500 LCt = 10,000 50 50 mg/man
35 Table 2-7. Dose-Related Effects of Sulfur Mustard on Skin at 16-27°C [30]
Latent Period Form Exposure Dosage Acute Signs and Symptoms Significance and Duration of Injury (hours)
Vapor 50 mg.min/m3 4-12 Mild erythema Threshold for nondisabling signs and symptoms
Injured but not disabled Extent of performance decrement dependent upon anatomical > 100-300 mg.min/m3 4-8 Erythema, itching, sensitivity to touch site and total area of skin surface affected Healing in 5-10 days Incapacitating injury Severe erythema, followed at 12-24 hr by Recovery period lasting several weeks to several months, ICt = 1,000-2,000 mg.min/m3 3-6 blistering, with new blisters continually 50 depending on anatomical site and total area of skin surface forming for 2-3 days affected
Rapid development of erythema, followed in Incapacitating injury for survivors LCt = 10,000 mg.min/m3 1-3 3-12 hr by severe blistering; concomitant 50 Prolonged hospitalization and convalescence likely systmic intoxication
Injured but not disabled Liquid 10-20 µg/cm2 1-3 Threshold dose for mild erythema Nondisabling signs and symptoms
36 Table 2-8. Dose-Related Effects of Sulfur Mustard on Eyes at 16-27°C [30]
Form Exposure Dosage Latent Period Acute Signs and Symptoms Significance and Duration of Injury
Several hours to Vapor ≤ 12 mg.min/m3 Reddening First indication of nondisabling signs and symptoms several days
Conjunctivitis, grittiness under eyelids, 50-100 mg.min/m3 4-12 hours Injured but not disabled for 2-7 days, 2 weeks in severe cases tearing, sensitivity to light
Corneal edema and clouding, eyelid edema, Incapacitating injury 3 ICt50 = 200 mg.min/m 3-12 hours photophbia, severe blepharospasm leading Healing progresses over several weeks to temporary blindess Hypersensitivity to mustard may last several months
Incapacitating injury Corneal damage with possible ulceration and Possible prolonged hospitalization, requiring several months to 400-800 mg.min/m3 1-4 hours secondary infection complete recovery Permanent eye damage in some cases
Severe corneal damage, possible loss of > 800 mg.min/m3 1-3 hours Same as above vision, possible systemic effects
Incapacitating injury Severe pain, edema, corneal damage; Liquid Unknown < 1 hour Hospitalization required possible corneal perforation In some cases permanent eye damage or loss of eye (rare)
37 Table 2-9. Dose-Related Effects of Sulfur Mustard on the Respiratory System at 16-27°C [30]
Exposure Dosage Latent Period Acute Signs and Symptoms Significance and Duration of Injury
Recovery period may last 2 weeks 12-70 mg.min/m3 12 hours - 2 days Irritation of nasal mucosa, hoarseness No significant performance decrement expected
Upper airway: Sneezing, lacrimation, rhinorrhea, Incapacitating injury ICt = 200 mg.min/m3 4-6 hours 50 epistaxis, sore throat, and hoarseness Recovery period about 2 weeks
Lower airway: Tracheobronchitis, hacking, cough, Prolonged recovery after secondary tachypnea, and pseudomembrane formation (may be infections accompanied by fever). Pulmonary edema and (1-2 months) bronchopneumonia may develop after 36-48 hours
Injury as described above, progressing to edematous changes in pharynx and tracheobronchial tree; possible Incapacitating injury for survivors LCt = 1,000-1,500 mg.min/m3 50 death due to secondary bacterial infections or necrotic Convalescence, several months bronchopneumonia
38 2.4 The Third Generation of Chemical Warfare Agents: Nerve Agents
2.4.1 Development of Organophosphorous Nerve Agents
At the close of World War I, the Allies forced Germany to surrender many of the territories
that it relied upon for grain production, and by 1933, it was highly dependent upon imports from foreign countries. Germany purchased approximately 30 million marks’ worth of insecticides in an attempt to increase domestic crop yields but was still dependent on foreign sources for the chemicals
[31]. Therefore the German chemical industry was called upon to develop better insecticides and end the dependence on foreign countries. In 1934, Dr. Gerhard Schrader was a chemist working for
Bayer Company, which was a subsidiary of the world’s largest corporation, IG Farben. Schrader’s chief area of research was on the development of the toxic chemicals for use as insecticides. After working on various fluorine-based compounds, Schrader shifted his focus to those based on sulfur, but neither group produced a viable insecticide. Moving across the periodic table, Schrader chose to experiment with phosphorous containing compounds. Upon initial investigation, the organophosphates exhibited sufficient insecticidal properties for IG Farben to procure patents in multiple countries. Schrader and his team investigated numerous organophosphorous-based molecules, and finally integrated the cyanide moiety into one of the variants. After working with the newly synthesized compound, Schrader experienced headaches, shortness of breath, and his pupils would not dilate. Schrader was forced to postpone his work for several weeks while he recovered from the exposure. On December 23, 1936, Schrader purified his compound and sent if off for toxicological testing. The testing showed that the new compound was extremely toxic to insects as exposure to 5 ppm killed 100% of lice on contact, and animals that were exposed to 25 mg/m3 were dead within twenty-five minutes [31]. Although the compound was too toxic to be used as an
39 insecticide, it was reported to the German military according to an ordinance that required any scientific finding with potential military use to be brought to the attention of German officials. After live demonstrations of the new lethal compound in action, some scientists suggested it was “taboo” or too strong. Thus, the new nerve agent was named Tabun after the German word for taboo (tabu) [31].
Germany quickly deemed this compound and all of its patents to be top secret and held any mention of the chemical out of scientific publications. The further development, pilot plant testing, and full- scale industrialization of Tabun was taken on by the German government. Schrader continued his work on insecticides, and in 1938 he synthesized another organophosphorous compound that contained fluorine. Once again, Schrader’s new compound was extremely toxic, reportedly two to ten times more potent than Tabun, and it was also considered classified by the German government. This new chemical agent was referred to as Sarin after the four members that were instrumental in its development: Schrader, Ambros, Rüdiger and Linde [4] [31]. Closer to the end of World War II,
Schrader developed another organophosphorous variant that was very similar to Sarin. With a much lower vapor pressure than Sarin, Cyclosarin was considered to be a persistent chemical agent.
German scientists believed that this property would allow Cyclosarin to be used in the same defensive or “area denial” techniques that was seen with mustard gas. The lower toxicity, costly manufacturing processes, and inflammability of the compound, along with the eminent close of World War II caused
Cyclosarin to be relatively ignored by the Germany military [4].
The last major nerve agent developed during World War II was synthesized in 1944 by Dr.
Richard Kuhn, the director of the Institute of Chemistry at the Kaiser Wilhelm Institute for Medical
Research [31]. Along with his research to investigate the effects of the nerve agents on the central and peripheral nervous systems, Kuhn synthesized a variant of Sarin by using pinacolyl alcohol as a starting material instead of isopropyl alcohol. This new agent, called Soman, was roughly two times
40 as potent as Sarin, and the ease with which it crossed the blood-brain barrier allowed for rapid onset of symptoms [31]. As with Cyclosarin, further development of Soman was hindered by a lack of resources. Throughout World War II, Germany produced massive amounts of Tabun and Sarin at specially designed manufacturing plants. Although stockpiles of the nerve agents were on hand,
Germany never used them on the battlefield. As mentioned previously, Adolf Hitler’s exposure to mustard gas in World War I may have affected his decision to not utilize Germany’s chemical weapons against the Allied forces, but there were other factors that also played into this decision. The
United States President, Franklin D. Roosevelt, stated that the U.S. would not “resort to chemical weapons unless they were used by the enemy first [31].” The promised heavy retaliation by the
Allied forces was most likely enough to deter Germany from unleashing the new nerve agents.
Even after the close of World War II, research continued on organophosphorous compounds for their potential insecticidal properties. At Imperial Chemical Industries (ICI) in United Kingdom two scientists, Ranajit Ghosh and J.F. Newman, synthesized an organophosphorous compound that incorporated a sulfur atom instead of the fluorine atom that was contained in Sarin and Soman. As with Schrader’s discoveries, this nerve agent was extremely toxic and unable to be used as an insecticide. The British government passed the development of the agent, which reportedly had fifty times the toxicity of Sarin, to the United States [4]. This new super-lethal class of agents became known as the “Venomous” class, with the primer agent named VX.
Toward the end of World War II, the various German nerve gases were discovered by the
Allied forces, and each of the agents was known by numerous names. A nomenclature was developed to eliminate any confusion when referring to the chemical weapons. The nerve agents developed in Germany comprised the G-series of agents. Since Tabun was the first nerve agent it was designated “GA”, Sarin was “GB”, Soman was “GD”, and Cyclosarin was “GF”. The code “GC”
41 was excluded because it could easily be confused with the designation for phosgene, “CG.”
Following the same fashion, the V-series of nerve agents included the later developed and more potent VX.
Although many countries stockpiled nerve agents after World War II and even into the Cold
War era, these sinister weapons were never released on the world in a large scale. One of the only documented uses of nerve agents in a war was in the 1980s during the Iran-Iraq War. In 1988, the
Iraqi leader, Saddam Hussein, approved the use of Sarin and mustard gas to destroy the Kurdish population in northern Iraq [31]. On March 13, 1988 the market town of Halabja was heavily bombed with artillery rounds followed by chemical agents. This attack resulted in an estimated 2,000 to 5,000 fatalities and another 10,000 casualties [31].
The main deterrent for chemical agent usage in war has always been the fear of retaliation.
However, terrorist groups have exploited the fact that there is no major concern for retaliation when unsuspecting and unprepared civilians are attacked. The Tokyo Subway Attacks in 1994 are an example of terrorist usage of chemical warfare agents. A Japanese religious cult released Sarin gas onto various subway trains resulting in 19 deaths and over 6,000 people seeking medical aid [32].
2.4.2 Nerve Agents: Physical and Chemical Properties
As a class of compounds, the organophosphorous nerve agents are all viscous liquids that vary greatly in their vapor pressures. As seen in Table 2-10, Sarin has the highest vapor pressure, and therefore it is not considered a persistent agent. Soman has the next highest vapor pressure followed by Tabun and Cyclosarin. The low vapor pressure of VX was one of the main properties that made it attractive for use as a chemical warfare agent. After an area is exposed to VX, the vapors can condense on the ground and persist for many days, making it a very effective terrain denial weapon.
42 Table 2-10. Physical and Chemical Properties of Organophosphorous Nerve Agents [33]
Agent GA GB GD GF VX
Common Name Tabun Sarin Soman Cyclosarin VX
Chemical Structure
Chemical C H N O P C H FO P C H FO P C H FO P C H NO PS Formula 5 11 2 2 4 10 2 7 16 2 7 14 2 11 26 2
Vapor Pressure 0.037 (20°C) 2.10 (20°C) 0.40 (25°C) 0.056 (20°C) 0.0007 (25°C) (mm Hg)
Volatility 610 22,000 3,900 817 10.5 (mg/m3 at 25°C)
Liquid Density 1.073 (25°C) 1.012 (20°C) 1.022 (25°C) 1.133 (20°C) 1.006 (20°C) (g/mL)
Freezing Point -50 -56 -42 -30 -39 (calculated) (°C)
Faintly fruity; no odor when Fruity, odor of camphor when Perceptible; fruity; Odor Odorless when pure Odorless when pure pure impure odorless when pure
43 Since VX has such a low vapor pressure, the most likely route of exposure to the agent is through skin contact. On the contrary, the other nerve agents are all capable of being generated in sufficiently lethal concentrated vapors. For that reason the primary area affected by Sarin, Tabun, Soman, and
Cyclosarin is the respiratory tract.
While the nerve agents were manufactured and stockpiled during the various wars, it was observed that some of them were not stable and therefore would breakdown during storage. After decomposition inside the artillery shell, the chemicals formed a thick sludge that altered the trajectory of the projectile. This inherent instability was not a desired property for an effective chemical warfare agent, and as a result, many countries sought to develop binary weapons that would combine two precursor compounds to form the nerve agent just after the projectile was fired [31].
2.4.3 Nerve Agents: Mechanism of Action
The term “nerve agent” is used to refer to the organophosphorous-based chemical weapons because of their significant effects on the body’s peripheral and central nervous systems. The specific mechanism of action involves the inhibition of the enzyme that breaks down the neurotransmitter acetylcholine (ACh). The two major branches of the body’s nervous system are the central nervous system (CNS), which is comprised of the brain and spinal column, and the peripheral nervous system
(PNS), which includes all the nerves in the rest of the body. The PNS is further divided into the somatic and autonomic nervous systems. The somatic nervous system is responsible for the voluntary movement of the skeletal muscles, and the autonomic nervous system controls the body’s visceral or instinctual processes such as heart rate, respiration, and digestion. The autonomic nervous system can be further divided into the sympathetic and parasympathetic systems, which control the
“Fight or Flight” and the “Rest and Digest” responses respectively [34]. With so many divisions of
44 the body’s nervous system controlling such a wide array of bodily functions it is easy to appreciate how one chemical agent can have such dramatic effects on an individual. Acetylcholine and its receptors can be found in all of the neuromuscular junctions in the nervous systems, and wherever this neurotransmitter is used the enzyme acetylcholinesterase (AChE) is also found. The mechanism for the normal activity of the esterase is shown at the top of Figure 2-9. The basic structure of the active position on the enzyme consists of the “anionic site” and the “esteratic site,” which contains the amino acid serine. Since the choline part of acetylcholine has a positively charged nitrogen atom, it is attracted to the “anionic site” and allows the neurotransmitter molecule to be held in place for the breakdown to occur. The carbonyl of the acetyl group is attacked by the serine residue and acetylcholine is broken down into choline and acetic acid or acetate. These compounds are recycled in the body and can be recombined to form acetylcholine that goes on to stimulate other receptors.
The enzyme allows this decomposition of acetylcholine to occur extremely fast so that the neurotransmitter cannot continue to stimulate receptors throughout the body. The nerve agents cause an irreversible inhibition of the enzyme, shown in Part b) of Figure 2-9, because they are capable of forming strongly covalent bonds between the phosphorous and serine residue. With the enzyme effectively inactivated by the bound nerve agent, an accumulation of acetylcholine occurs which causes the nerve receptors to be constantly stimulated. Depending on the branch of the PNS and which receptors are activated, the symptoms can ranged from muscle fasciculations and paralysis to miosis, bronchoconstriction, abdominal cramps, and salivation [33]. The effects of exposure to nerve agents on the CNS can consist of confusion, anxiety, fatigue, slurred speech, and death [33].
A main area of research for all countries that developed nerve agents was the exploration and synthesis of chemicals that could potentially serve as antidotes to reactivate the acetylcholinesterase.
45
Figure 2-9. Mechanism of Acetylcholinesterase Activity, Inhibition, and Reactivation
46 Atropine, extracted from the deadly nightshade plant, was known to produce effects opposite to those experienced from nerve agent exposure, such as dilating the pupils and increasing the heart rate [31].
During his research to develop Soman, Dr. Richard Kuhn also sought for a cause for such wide ranging effects on the body’s nervous systems. Through his work, Kuhn discovered the importance of acetylcholine and each of the receptors that it affects. Kuhn’s team developed an assay to quickly determine the effectiveness of new nerve agents at inhibiting acetylcholinesterase. The testing of
Soman showed that it irreversibly inactivated the enzyme within two minutes and severely lessened the effectiveness of atropine as an antidote. It was later discovered by Dr. David Nachmansohn at
Colombia University that atropine only blocked the receptors for acetylcholine instead of reactivating the enzyme [31]. Further research showed that the compound, pyridine aldoximine methiodide
(PAM) was capable of removing the nerve agent molecule from the enzyme resulting in its reactivation. With the two antidotes administered simultaneously, PAM reactivated the enzyme while atropine blocked the receptors providing a ten-fold increase in the dose of VX needed for lethal effects [31]. This mechanism is given in Part c) of Figure 2-9. Upon this discovery, soldiers were supplied with automatic injection syringes filled with the antidotes in the event that they were exposed to any of the nerve agents.
47 CHAPTER 3: Modern Chemical Protective Ensembles
3.1 Introduction to Modern Chemical Protective Equipment
3.1.1 Need for Personal Protective Equipment for Specific Routes of Exposure
Many advances in the field of personal protective equipment (PPE) have been made since the
first respirators were developed after Germany’s chlorine attacks. The arrival of mustard gas and its blistering ability in the latter stages of World War I made respirators an insufficient means of protection. Instead of only protecting the eyes and respiratory tract, the entire body surface had to be separated from the contaminated environment. As history has shown, the susceptibility of the various routes of exposure and the development of PPE have tracked closely alongside one another. As irritant gases were deployed to attack the highly sensitive respiratory tract, respiratory protection was developed. Tear gases were then utilized to attack the eyes, causing soldiers to remove their gas masks, and ultimately expose the respiratory tract to the warfare agent. To defeat this tactic and protect the eyes, tight fitting goggles or full face respirators were invented. Finally, total body protection and full ensembles were developed to provide an extra barrier between the environment and the skin. One reason that the lungs and respiratory tract are so vulnerable is because of the thin membrane between the inhaled air and the blood stream. Another significant factor is the large surface area compared to the other routes of exposure. While eye injuries can be disabling, the overall surface area of the eye is only 0.0002 m2, whereas the surface area of the skin of an adult can
cover 1-2 m2, and the surface area of the lungs of an average adult human can be 50-100 m2 [35].
Therefore the routes of exposure listed by importance for protection and susceptibility to injury are:
1) vapor exposure to the respiratory tract, 2) liquid exposure to the skin, and 3) vapor exposure to the skin [35].
48 3.2 Modern Respiratory Protection
3.2.1 Overview of Different Levels and Types of Respiratory Protection
Since the respiratory tract is the most susceptible route of exposure and can result in rapid
systemic contamination, it is understandable that developing a means of respiratory protection was
the first response to the chemical agent attacks in the First World War. Modern respirator
technologies are no longer solely used for the military to prevent exposure to CWAs, instead they are
vital in protecting the firefighters and other emergency responders that protect and rescue civilians
each and every day. Respirators are also used in many industrial settings to limit workers’ exposure
to toxic industrial chemicals (TICs) that can be extremely hazardous in their own right.
As is the case with all protective clothing, the need for balance between protection and
usability is a key component in the design of respirators. Although the main goal is to provide a
barrier to the toxic atmosphere, it is important that the respirator does not add a high degree of
physiological or psychological burden to the wearer, and it must also not interfere with the
individual’s tasks or other equipment that is being used. The two major issues that arise in respirator
design are that there is not enough space for all of the components that are needed and anything that is
done to improve one facet of the design will most likely take away from another area [36]. Therefore,
the ideal respirator would provide the best protection possible while allowing a high degree of
comfort and no hindrance to the tasks that must be performed.
Current respiratory protection can be categorized as either atmosphere (air)-supplied
respirators or air-purifying respirators. Each type of respirator has its own advantages and
disadvantages, but if used according to regulatory guidelines, the user should be well protected from
exposure using either type. The key feature of the atmosphere-supplied respirator is that it provides the user with a clean source of breathing air that is entirely separated from the contaminated
49 environment. This type of respirator provides the best protection to the respiratory tract and is
designated as the protection of choice when the individual is exposed to a threat of unknown identity
or concentration. One of the most common types of atmosphere-supplied regulators is the self- contained breathing apparatus (SCBA) that is used by firefighters and other emergency responders around the world. In the case of the SCBA (Figure 3-1), the clean source of air is held in a gas cylinder that is worn on the back of the individual. The air tank provides only thirty to sixty minutes of clean air, and it also adds a physiological burden of carrying the extra weight [35].
Figure 3-1. Self-Contained Breathing Apparatus from MSA [37]
50 Another form of atmosphere-supplied respirator incorporates a hose up to 300 feet in length that is connected to a group of large stationary air tanks or an air compressor equipped with a capable filter [35]. Although this method allows for the individual to remain in the contaminated environment for longer periods of time (compared to the SCBA), there is the potential that the hose could become kinked, knotted, or even punctured. The photo on the left in Figure 3-2 shows how the SCBA is worn by an emergency responder, and the photo on the right shows a variation with the air tank on the hip.
Figure 3-2. Various Atmosphere-Supplied Respirators [38] [39]
51 The protective masks developed during World War I all fit into the air-purifying respirator category. The main component of this type of respirator is the filter cartridge that is positioned between the contaminated environment and the individual’s respiratory tract. Upon inhalation, the contaminated air is drawn through the filter where the toxic chemicals can be neutralized or sequestered by the filter medium. Since its first use in the German gas masks and the Large Box respirators, activated carbon has become the most frequently used sorbent in the respirator cartridges.
Modern cartridges are filled with activated carbon to trap the chemical vapors but the carbon is also impregnated with copper, silver, zinc, and molybdenum salts as well as triethylenediamine to increase the efficacy of the purification process [35]. To aid in the protection against solid particles such as soot, dust, ash, or the arsenic-based sneezing agents, a particulate filter material can also be included.
A cross-sectional view of an average military filter is shown in Figure 3-3.
Figure 3-3. Cross-sectional View of Respirator Cartridge [36]
52 The carbon is activated to increase its capacity to trap chemical vapors by maximizing the surface area available for the adsorption process. A normal chemical warfare cartridge can be filled with approximately 200 grams of activated carbon which can have adsorptive surface areas of 500-
1,400 m2 per gram. Therefore, one filter cartridge can have approximately 35 times the surface area of a regulation size football field (approximately 5,300 m2) [35]. This large surface area allows the individual to breathe through the filter for much longer than the thirty to sixty minutes allowed by an average SCBA. The extended period of usage is one of the reasons that the air-purifying respirators like the one shown on the right of Figure 3-5 are preferred over the atmosphere-supplied respirators.
They are small, compact, lightweight, and do not need a large air tank that must be refilled. However, there are guidelines set forth by the Occupational Safety and Health Administration (OSHA) that regulate the usage of the air-purifying respirator. These regulations state that this means of protection can only be used if the toxic chemical has been identified and its concentration is below that which is considered immediately dangerous to life or health (the IDLH value) [35]. Also air-purifying respirators, regardless of design, cannot be used in an environment that is oxygen-deficient (below
19.5% in air). These OSHA regulations mainly apply to and govern the industrial sector to limit the exposure of civilians. Due to the highly trained nature of the soldiers and the need for lightweight protection, the military does not follow these regulations [35]. The IDLH values for common chemical warfare agents are given in Table 3-1. As would be expected, the hazardous nerve agents have very low IDLH values.
53 Table 3-1. IDLH Values for Common Chemical Warfare Agents
Chemical Agent Acronym IDLH in ppm IDLH in mg/m3 Molecular Weights
Tabun GA 0.02 0.1a 162.13 Sarin GB 0.02 0.1a 140.10 Soman GD 0.007 0.05a 182.18 - VX 0.0003 0.003a 267.38 Mustard HD 0.11 0.7a 159.08 Cyanide AC 50 60b 27.03 Phosgene CG 2 10b 98.92 b Chlorine Cl2 10 30 70.91 b Ammonia NH3 300 230 17.03
a: taken from [40] b: taken from [35]
There are many different designs of air-purifying respirators, but the main variable is whether the face piece is held in positive or negative pressure. Negative-pressure full-faced air-purifying respirators like the one shown in Figure 3-4 are more universally known because they are used in many different applications, and they are very simple for the average user to operate effectively.
These respirators are lightweight, easy to maintain, have a limited effect on mobility, and are one of the least expensive to use [35]. The positive-pressure respirators are known as powered air-purifying respirators (PAPR), and as seen in Figure 3-4, they incorporate a battery-powered blower that forces air into the face piece. The advantage of the blower is that it creates a positive pressure inside the face piece so that if the seal with the face was to be compromised, the positive pressure would not allow any chemical agent vapors to enter the respirator [35].
54
Figure 3-4. Different Designs of Air-Purifying Respirators [41]
55 Air-purifying respirators can have many different designs based on the various end uses. An example can be seen by the differences in the two full-face respirators shown in Figure 3-5. The mask on the left is used for industrial applications and has two separate respirator cartridges. Having two filter cartridges increases the adsorptive capacity for toxicants, allows the individual to wear the respirator for longer periods, and lessens the restriction on breathing that is inherent with all respirator usage. The mask on the right is the military M40 respirator, and it only has one filter cartridge because it is imperative that the design of the respirator does not interfere with the soldier’s ability to aim and fire weapons [36]. Having a filter cartridge on both sides of the mask would either hinder this ability or cause a disruption to the face piece seal.
Figure 3-5. Full-Face Air Purifying Respirators: Industrial [42] and Military [43]
56 The M40 respirator is currently being replaced by the newly designed M50 Joint Service
General Purpose Mask (JSGPM) (Figure 3-6) manufactured by Avon Protection Systems, Inc. The
M50 does incorporate two lower profile filters with shut-off valves so that the individual can change the cartridges without having to remove the mask and has been designed to be more comfortable than
its predecessor. As shown in Figure 3-6, the soldiers are taught to hold their heads at a steeper angle
and to tilt the weapon to avoid any issues with the filters. This evolution from the M40 to the M50
respirator is a perfect example of the constant balancing act between comfort, protection, and
functionality with which the respirator designers are faced. The M50 respirator is designed to be
more comfortable, reduce the breathing resistance, and provide longer periods of protection, but these
advantages come at the cost of having to alter the way that the soldiers aim their weapons.
Figure 3-6. M50 JSGPM [41] and Effect of Cartridge on Aiming of Weapon [44]
57 Aside from the filter cartridges, there are many more devices and technologies that must be incorporated into an effective respirator. The inhalation and exhalation valves along with the nosecup are designed to regulate and direct the airflow inside the face piece so that the air flows over the eyepieces, reducing the potential for fogging of the lenses. The nosecup also limits the dead volume of air or the volume of air that must be displaced from the respiratory tract during respiration [35].
Modules such as voicemitters and amplifiers are included to increase the clarity of the individual’s communications. Drinking tubes and adapters can also be incorporated inside the face piece to allow the user to rehydrate in a contaminated environment without removing the protective equipment.
With all of the devices that must be included in the mask, the issue of space is a constant factor in respirator design. This issue is magnified when manufacturing smaller sized respirators because the space inside the face piece is decreased but all of the devices and valves remain the same size [36].
Also making sure that the respirator will fit each of the possible sizes and shapes of heads is extremely difficult because most respirators are only manufactured in four sizes (extra-large, large, medium, and small). Although respirator technology has advanced significantly since its initial introduction to the battlefield, there are still many areas that can be improved as respirators become more protective, comfortable, better fitting, and more efficient.
3.3 Modern Total Body Protection
3.3.1 Guidelines for Selection of Appropriate Level of Chemical Protection
Germany’s introduction of mustard gas during World War I completely changed the direction of the chemical protection industry. Although protecting the respiratory tract was the first priority, materials and ensembles had to be developed to protect the entire body from exposure to vesicating
58 agents. Some of the first total body chemical protective clothing ensembles were constructed out of rubberized fabric which was impermeable to the warfare agents, but it also did not allow any transfer of moisture from the evaporated sweat out of the garment. Since these fabrics held in the heat and the moisture they could only be worn for short periods of time and many soldiers chose not to use them
[4]. The designers of this first chemical protective clothing quickly realized the importance of creating a balance between protection and comfort. An ensemble can be completely impervious to the potential threat, but the soldier may only be able to work in the ensemble for short periods of time before suffering extreme levels of heat stress. Therefore, it is imperative to match the potential threat or exposure with the appropriate level of protection. Basically, modern chemical protective clothing is specifically designed for different threat levels, and the selection of the appropriate ensemble is of utmost importance.
To aid in the selection process, the United States Environmental Protection Agency (EPA) has defined four separate levels (A, B, C, and D) of protection outlined by the Code of Federal
Regulations 29 CFR 1910.120 [35]. Examples of each level of protection and their distinguishing characteristics are given in Table 3-2. The specific level of protection that is required depends on the chemical or agent that is present, the toxicity of the agent, the exposure concentration, type of exposure, and duration of the exposure [35] [45]. Chemical protective clothing designated as Level A provides the highest level of both respiratory and dermal protection. The regulations specify that
Level A suits must fully encapsulate the individual and all equipment including the mandatory SCBA respirator. This level of protection is specifically used if the chemical agent or threat is unknown and/or at extremely high concentrations. All of the seals, seams, and zippers incorporated into Level
A ensembles must be impermeable and provide an air-tight closure. Since Level A suits are completely impermeable they provide the highest level of protection but also the highest risk of heat
59 stress and exhaustion [35]. In order to select a level of protection lower than Level A, a risk assessment must be carried out so that the chemical identity and concentration are known.
Table 3-2. EPA Levels of Chemical Protective Equipment [35] [45] [46]
EPA Level Level Aa Level Ba Level Ca Level Da
Example of Suit Type
Vapor Protection Yes No No No
Liquid Protection Yes Yes Yes No
Type of Respiratory Air-Purifying SCBA SCBA None Protection Respirator
Dupont™ Dupont™ Dupont™ Dupont™ Suit Material Tychem ® BR Tychem ® BR Tychem ® BR Tychem ® BR
Maximum level of Same as Level B, but Liquid splash Comments protection from both different respiratory Basic work uniform protection only vapor and liquid protection level
a: All photos taken from [46]
60 The second level of protection, Level B, is recommended when there is a low chance of exposure through the dermal route, but the respiratory tract must still be protected at the highest level.
Level B suits can look exactly like Level A suits but the seams, seals, and zippers are no longer required to be air-tight. Whereas Level A suits must be one piece, Level B suits can come in three different forms: a one-piece encapsulating suit, a one-piece coverall with an open face, or a two-piece coverall with open face [35]. By stepping down from a Level A to a Level B ensemble, the individual loses almost all dermal protection against vapor exposures but still maintains protection against liquid splashes. The SCBA respirator is still required for this level of protection, but can be worn on the outside of the garment. Both Level A and Level B chemical protective clothing must be worn in oxygen-deficient environments.
The only difference between Level B garments and Level C garments is the degree of respiratory protection. Level C garments still provide the same amount of liquid splash protection, but air-purifying respirators can be used instead of an SCBA. One of the most common Level C ensembles is the Joint Service Lightweight Integrated Suit Technology (JSLIST) used by the military.
This ensemble, shown in Figure 3-7, consists of a two-piece overgarment that can be worn as a primary uniform. The JSLIST is a two-layer fabric system with the outer layer consisting of a 50/50 nylon/cotton poplin ripstop fabric finished with a durable water repellent, and the inner liner is made of a nonwoven material laminated with activated carbon to actively adsorb chemical like the filters in the respirators [47]. The JSLIST ensemble also incorporates chemical protective gloves, over boots, and an air-purifying respirator like the M40 or M50 JSGPM. Since the military has needs that are specific to soldiers that are out on the battlefield for long periods of time, the JSLIST is capable of being laundered up to six times [47].
61 The lowest protection level according the EPA scale is Level D. This type of ensemble is essentially any standard work uniform and does not provide any measurable protection for the respiratory tract or skin. Basic latex gloves, surgical masks, gowns, or aprons that are used by medical personnel fall into the Level D category [35].
Figure 3-7. Joint Service Lightweight Integrated Suit Technology (JSLIST) [48] [49]
62 3.3.2 Performance Criteria for Chemical Protective Ensembles
The EPA levels of protection were initially developed to address the need for different
degrees of full body protective clothing for workers on hazardous materials cleanup sites. The main
goal of the guidelines is to specify the design requirements that are needed for each level, such as
fully encapsulating suits for Level A ensembles. Although the EPA levels are a good starting point
for determining what type of suit should be worn for a specific threat, they do not specify any
performance criteria for the different parts of the ensemble. In other words, there are no definitions or
minimum requirements for terms such as chemically resistant or totally encapsulating [45]. To
address this issue, in 1986 the National Fire Protection Association (NFPA) appointed committees to
develop performance-based standards for both vapor-protective and liquid splash-protective ensembles [45]. As a result of their efforts, the NFPA released the following two standards:
• NFPA 1991: Standard on Vapor-Protective Ensembles for Hazardous Materials Emergencies
• NFPA 1992: Standard on Liquid Splash-Protective Ensembles and Clothing for Hazardous
Materials Emergencies
The NFPA 1991 Standard sets the performance criteria for a Level A equivalent ensemble that provides the highest level of both vapor and liquid protection. The performance criteria for both
Level B and Level C ensembles for only liquid splash protection are stated by the NFPA 1992
Standard. One section of the standards specifically states the minimum requirements for all parts of a
chemical protective ensemble including the suit material, seams, closures, visors, gloves, and
footwear. A separate section explains how each test should be carried out and references any other
standard methods that should be followed. Generally, the NFPA revises the standards on a five-year
cycle, and since their introduction in 1991, these standards have been revised three times with a
fourth revision due out in 2012. The performance criteria for the 2005 Edition of both the NFPA
63 1991 and 1992 standards for key elements of the ensemble are given in Table 3-3. Examples of
ensembles commercially available from Dupont ™ that are certified to NFPA 1991 and NFPA 1992
are given in Table 3-4 and Table 3-5, respectively.
In 1998, the NFPA began working on a new standard to establish performance criteria for the
protective ensembles of first responders at the scene of a terrorist incident involving Chemical,
Biological, Radioactive, and Nuclear (CBRN) agents [50]. The sole focus of this standard was to
provide the appropriate level of protection from CBRN agents while still allowing the emergency
personnel to rescue victims and provide medical attention to exposed individuals.
Just over one month before the terrorist attacks at the World Trade Center on September 11,
2001, NFPA 1994: Standard on Protective Ensembles for First Responders to CBRN Terrorism
Incidents was released [45]. The attacks on that day changed the way the world viewed chemical
protective clothing. Up until that point, most of the chemical protective ensembles were either for
workers in the chemical industry, hazardous materials cleanup teams, or the military. From that point
on it became apparent that providing appropriate CBRN protection for the firefighters, policemen,
medical personnel, and other first responders on the scene of a terrorist attack was a critical need of
the highest priority. Whereas the NFPA 1991 and 1992 standards were designed for hazardous
materials response and management, the requirements in NFPA 1994 were specifically set according
to the tasks and responsibilities of first responders [51]. The first edition of the standard created three
separate classes of ensembles, Class 1, Class 2, and Class 3. The Class 1 ensembles matched closely
with the Level A garments in that they provided the highest level of vapor and liquid protection.
Class 2 ensembles provided a limited amount of vapor protection with a high level of liquid-splash protection, and the Class 3 ensembles, like the Level C garments, were used only for liquid-splash
64 protection. During the revision cycle, the Class 1 ensembles were absorbed into the 2005 Edition of
the NFPA 1991 Standard [50].
Since the chemical resistance testing in NFPA 1991 uses a full battery of TICs and CWAs at high concentrations to simulate the worst case exposures, Class 1 ensembles must be certified to the these requirements to ensure that they provide the absolute highest level of protection. Therefore the
2007 Edition of NFPA 1994 describes Class 2, Class 3, and Class 4 ensembles. Some of the important performance criteria for each ensemble class are provided in Table 3-6.
As with the first version of the standard, the Class 2 ensembles provide limited vapor and liquid protection, and they must be equipped with an SCBA because they are designed to be worn in the “Hot Zone” after a CBRN incident. The “Hot Zone” is the closest to the incident, contains agents or chemicals at or above the IDLH levels, and provides the highest risk of exposure. An example of a
Class 2 ensemble is shown in Table 3-7. This ensemble is very similar to the NFPA 1991 certified ensemble shown in Table 3-4, and is actually worn underneath the aluminized over suit.
Class 3 ensembles provide a lower level of protection because they are designed to be used in the “Warm Zone” where hazards are only present because of interaction with contaminated individuals or equipment. The concentration of any agent or chemical should be below the IDLH value and permits the use of an air-purifying respirator. The performance criteria for Class 3 ensembles are therefore lower than those of the Class 2 ensembles. Various examples of Class 2 and
Class 3 ensembles commercially available from Lion® Apparel and Blauer® are given in Table 3-8.
The Class 4 ensembles are only designed to provide protection against biological or radioactive particle hazards and are not intended for protection against chemical hazards [51]. Out of the three classes, the Class 4 ensembles provide the lowest level of protection, and should only be used in the “Cold Zone.”
65 Table 3-3. Overview of Key NFPA 1991 and 1992 Performance Criteria [45] [52] [53]
Test Method/ NFPA 1991 NFPA 1992 Property Measurement Vapor-Protective Liquid Splash-Protective
Liquid-tight integrity (suits) ASTM F 1359 No leakage No leakage
Permeation resistance of Breakthrough times ≥ 1 hour primary materials and seams ASTM F 739 Permeation rate ≤ 0.10 µg/cm2/min - (industrial chemicals) (all chemicals tested at full strength)
≤ 4.0 µg/cm2/ hour for HD Permeation resistance of ASTM F 739 ≤ 1.25 µg/cm2/ hour for GD primary materials and seams - (modified) 2 (chemical warfare agents) Liquids at 100 g/m Closed-top cell configuration
Penetration resistantance of ASTM F 903 - Penetration times > 1 hour primary materials and seams
Not ignite during 3-second exposure Burn distance ≤ 100 mm (4 in.) Flammability resistance ASTM F 1358 - Not sustain burning > 10 seconds Not melt during 12-second exposure
Suit and visor burst strength ASTM D 751 ≥ 200 N (45 lbf) ≥ 135 N (30 lbf)
Suit and visor puncture ASTM D 2582 ≥ 49 N (11 lb ) ≥ 25 N (5.6 lb ) propagation tear resistance f f
Suit and visor seam or ≥ 2.88 kN/m ≥ 67 N/50mm ASTM D 751 closure strength (30 lbf/2 in) (15 lbf/2 in) Cut resistance ≥ 25 mm (1 in) under ≥ 25 mm (1 in) under ASTM F 1790 (gloves) force of 200 grams force of 75 grams
Puncture resistance ASTM F 1342 ≥ 22 N (5 lb ) ≥ 11 N (2.5 lb ) (gloves) f f
Average percent increase Average percent increase Glove dexterity ASTM F 2101 ≤ 600% ≤ 200%
Cut resistance ≥ 25 mm (1 in) under ≥ 25 mm (1 in) under ASTM F 1790 (footwear upper) force of 400 grams force of 400 grams
Puncture resistance ASTM F 1342 ≥ 36 N (8 lb ) ≥ 36 N (8 lb ) (footwear upper) f f
Abrasion resistance ASTM D 1630 Abrasion rating ≥ 65 Abrasion rating ≥ 65 (footwear sole)
Slip resistance Coefficent of friction Coefficent of friction ASTM F 489 (footwear sole) ≥ 0.75 ≥ 0.75
66 Table 3-4. Example of DuPont™ NFPA 1991 Vapor-Protective Ensemble [46]
Back View Left Side View Front Side Right Side View
DuPont ™ Tychem ® TK Ensemble Identification (combined with aluminized fiberglass outersuit) EPA Protection Level Level A
NFPA Certification NFPA 1991 Ensemble
67 Table 3-5. Example of DuPont™ NFPA 1992 Liquid Splash-Protective Ensemble [46]
Back View Left Side View Front View Right Side View
Ensemble Identification DuPont ™ Tychem ® ThermPro
EPA Protection Level Level B (with SCBA) or Level C (with air-purifying respirator)
NFPA Certification NFPA 1992 Ensemble
68 Table 3-6. Overview of Key NFPA 1994 Performance Criteria [45] [50]
Test Method/ NFPA 1994 NFPA 1994 NFPA 1994 Property Measurement Class 2 Class 3 Class 4 Overall particulate inward NFPA 1994 8.5 - - ≤ 5% leakage
Man-In- PPDFi ≥ 360 PPDFi ≥ 120 Inward gas leakage - Simulant-Test PPDFsys ≥ 361 PPDFsys ≥ 76
Liquid-tight integrity (suits) ASTM F 1359 No leakage after 20 minutes No leakage after 4 minutes -
Liquid-tight integrity ASTM D 5151 No leakage No leakage No leakage (gloves and footwear) (modified)
Breakthrough times ≥ 1 hour Breakthrough times ≥ 1 hour Permeation resistance of Gases at 350 ppm Gases at 40 ppm primary materials and seams ASTM F 739 - Liquids at 10 g/m2 Liquids at 10 g/m2 (industrial chemicals) Closed-top cell configuration Open-top cell configuration
≤ 4.0 µg/cm2/ hour for HD ≤ 4.0 µg/cm2/ hour for HD Permeation resistance of ASTM F 739 2/ hour for GD 2/ hour for GD primary materials and seams ≤ 1.25 µg/cm ≤ 1.25 µg/cm - (modified) 2 2 (chemical warfare agents) Liquids at 10 g/m Liquids at 10 g/m Closed-top cell configuration Open-top cell configuration Biopenetration resistance of None after flexing and ASTM F 1671 None after flexing and abrasion None after flexing and abrasion primary materials and seams abrasion
Suit and visor burst strength ASTM D 751 ≥ 156 N (35 lbf) ≥ 135 N (30 lbf) ≥ 135 N (30 lbf)
Suit and visor puncture ASTM D 2582 ≥ 31 N (7 lb ) ≥ 25 N (5.6 lb ) ≥ 25 N (5.6 lb ) propagation tear resistance f f f
Suit and visor seam or ≥ 1.31 kN/m ≥ 1.31 kN/m ≥ 1.31 kN/m ASTM 751 closure strength (30 lbf/2 in) (15 lbf/2 in) (30 lbf/2 in) Average percent increase Average percent increase Average percent increase Glove dexterity ASTM F 2101 ≤ 300% ≤ 200% ≤ 200%
Cut resistance ≥ 25 mm (1 in) under ≥ 25 mm (1 in) under ≥ 25 mm (1 in) under ASTM F 1790 (gloves) force of 200 grams force of 100 grams force of 100 grams
Cut resistance ≥ 25 mm (1 in) under ≥ 25 mm (1 in) under ≥ 25 mm (1 in) under ASTM F 1790 (footwear upper) force of 600 grams force of 400 grams force of 400 grams
Puncture resistance ASTM F 1342 ≥ 17 N (3.8 lb ) ≥ 11 N (2.5 lb ) ≥ 11 N (2.5 lb ) (gloves) f f f
Puncture resistance ASTM F 1342 ≥ 36 N (8 lb ) ≥ 36 N (8 lb ) ≥ 36 N (8 lb ) (footwear upper) f f f
Abrasion resistance ASTM D 1630 Abrasion rating ≥ 65 Abrasion rating ≥ 65 Abrasion rating ≥ 65 (footwear sole)
Slip resistance Coefficent of friction Coefficent of friction Coefficent of friction ASTM F 489 (footwear sole) ≥ 0.75 ≥ 0.75 ≥ 0.75
69 Table 3-7. Example of DuPont™ NFPA 1994 Class 2 Ensemble [46]
Back View Left Side View Front View Right Side View
Ensemble Identification DuPont ™ Tychem ® TK
EPA Protection Level Level A
NFPA Certification NFPA 1994 Class 2 Ensemble
70 Table 3-8. Various Chemical Protective Ensembles from Lion® Apparel and Blauer® [54] [55]
Blauer ® HZ9420 Blauer ® Blauer ® Lion® MT94 LION® ICG LION ® MIGZ3 Multi-Threat Ensemble WZ9430 MIRT WZ9435 XRT
EPA Protection Level
Level B Level B Level C Level B Level C Level C
NFPA Certification
NFPA 1992 NFPA 1992 NFPA 1992 NFPA 1994 Class 3 NFPA 1994 Class 3 NFPA 1994 Class 3 NFPA 1994 Class 2 NFPA 1994 Class 2 NFPA 1994 Class 2
71 3.4 Standard Test Methods for Evaluating Chemical Protective Materials
3.4.1 Introduction to Material Level Chemical Testing
One of the first steps in the evaluation of chemical protective ensembles is material level
testing. Before any material or fabric can be incorporated into a NFPA certified ensemble it must
prove to perform at or above the required criteria for the specific standard. The type of testing that is
required is completely dependent upon the end use of the ensemble. The ensembles certified to
NFPA 1991 and NFPA 1994 must be constructed from materials that have shown to be resistant to
chemical permeation due to the high degree of vapor protection that is required. In contrast, the
liquid splash-protective ensembles certified to NFPA 1992 do not provide vapor protection, and therefore, they are mainly tested for liquid penetration. The terms ‘penetration’ and ‘permeation’ can often be confused and are commonly used interchangeably, but there is a distinct difference between the two processes that these terms describe. The process of chemical penetration can be defined as movement of the chemical through the protective material by means of defects in the surface, through seams, zippers, or other closures on a non-molecular level [35] [56]. Alternatively, the process of chemical permeation involves the movement of a chemical through a material on the molecular level whereby individual molecules of the chemical pass around or between the molecules of the material
[35] [56]. Since permeation occurs on the molecular level, it provides a means to test the resistance of chemical vapor transport through a material, whereas penetration testing provides useful information about the liquid protection capacity of a material.
72 3.4.2 Chemical Permeation Test Method and Battery of Chemicals
According to the NFPA standards, all materials that are incorporated into vapor-protective
ensembles must be tested for their resistance to chemical permeation against a range of TICs and
CWAs [52]. The battery of chemicals used for both NFPA 1991 and NFPA 1994 certification testing
are listed in Table 3-9 with their appropriate concentrations and physical states. Since NFPA 1991
governs the highest level of protection it incorporates a larger battery of chemicals at higher
concentrations than the NFPA 1994 requirements. The table also shows that Class 2 ensembles are
exposed to higher concentrations of gaseous chemicals than the lower protective Class 3 ensembles.
However, since both classes of ensembles are required to provide the same level of liquid splash- protection, they are exposed to the same level of liquid challenge.
The NFPA standards require that all permeation testing basically follow the standard test methods of the American Society for Testing and Materials (ASTM), specifically ASTM F 739:
Standard Test Method for Permeation of Liquids and Gases through Protective Clothing Materials under Conditions of Continuous Contact [56]. The newest version of the permeation test cell that is suggested in the NFPA standards is shown in Figure 3-8. The material is placed between the two sides of the cell with the challenge side facing up. A 1.0 L/min flow of clean, filtered air passes through the bottom of the cell and sweeps the underside of the material [52]. Any chemical that permeates through the material will be collected by an adsorbent, a solvent bubbler, or sent straight to an analyzer. The time that is required for the chemical to permeate the material and the rate of permeation are reported. The material is exposed to the chemical challenge for 1 hour (NFPA 1994) or 3 hours (NFPA 1991) depending on the specific standard. The performance criteria (Table 3-4 and
Table 3-6) can either be based on a cumulative permeation rate or on the breakthrough time.
73 Table 3-9. Concentration or Liquid Density of Permeation Test Chemicals [50] [52]
NFPA 1994 NFPA 1994 Chemical Liquid Gas NFPA 1991 Class 2 Class 3
Toxic Industrial Chemicals
Acrolein X 10 g/m2 10 g/m2 Acrylonitrile X 10 g/m2 10 g/m2 Acetone X 100 g/m2 Acetonitrile X 100 g/m2 Carbon disulfide X 100 g/m2 Dichloromethane X 100 g/m2 Diethyl amine X 100 g/m2 Dimethyl formamide X 100 g/m2 Ethyl acetate X 100 g/m2 Hexane X 100 g/m2 Methanol X 100 g/m2 Nitrobenzene X 100 g/m2 Sodim hydroxide X 100 g/m2 Sulfuric acid X 100 g/m2 Tetrachloroethylene X 100 g/m2 Tetrahydrofuran X 100 g/m2 Toluene X 100 g/m2 Ammonia X Pure 350 ppm 40 ppm 1,3-Butadiene X Pure Chlorine X Pure 350 ppm 40 ppm Ethylene oxide X Pure
Hydrogen chloride X Pure
Methyl chloride X Pure Chemical Warfare Agents Sarin (GB) X 100 g/m2 Soman (GD) X 10 g/m2 10 g/m2 Sulfur Mustard (HD) X 100 g/m2 10 g/m2 10 g/m2 Dimethyl sulfate X 100 g/m2 10 g/m2 10 g/m2 Cyanogen chloride X Pure Carbonyl chloride X Pure Hydrogen cyanide X Pure
74
Figure 3-8. NFPA 1991 and NFPA 1994 Permeation Test Cell
3.4.3 Test Methods for Resistance to Chemical Penetration
Whereas the NFPA 1991 and NFPA 1994 standards reference ASTM F 739 for permeation testing, NFPA 1992 references ASTM F 903: Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Liquids [53]. In this test method, the material is placed in the cell (Figure 3-9), the assembled cell is then connected to the pressure apparatus (Figure 3-9), and then the challenge liquid is injected into the cell. The standard has different procedures that can be followed that vary the time before and after application of pressure as well as the amount of pressure and time applied. The chemical battery for NFPA 1992 is much smaller than that of the other standards. The chemicals for penetration testing include: acetone, dimethyl formamide, ethyl acetate, nitrobenzene, tetrahydrofuran, 50% (w/w) sodium hydroxide, and 93.1% (w/w) sulfuric acid. The results of penetration testing are simply a ‘Pass’ is no chemical penetration is present over the entire test duration or a ‘Fail’ if any chemical penetration is observed [57].
75
Figure 3-9. ASTM F 903 Penetration Test Cell and Test Apparatus [57]
76 CHAPTER 4: Man-In-Simulant-Test: Background, Protocols, and Analysis
4.1 Introduction to Man-In-Simulant-Test (MIST)
4.1.1 Background and Basic MIST Methodology
Penetration and permeation testing can provide important information about the materials of which chemical protective ensembles are constructed, but the data are limited to only describing a flat piece of the material. More extensive and more specific testing is required to evaluate the entire chemical protective ensemble as it fits on and around the individual’s body. Instead of being a single, continuous, flat layer surrounding the body, the materials used in chemical protective ensembles contain seams, zippers, closures, and other interfaces. Therefore an ensemble constructed of even the most impermeable material can provide very low vapor or liquid protection if these interfaces are not equally as impermeable. To address the issue of evaluating protective performance of full-ensembles, a task group of the U.S. Army Chemical and Biological Defense Command (CBDCOM) developed the Man-In-Simulant-Test (MIST) in the mid-1990s [58]. The main principle behind the MIST is to expose human test subjects, dressed in full chemical protective ensembles, to a chemical warfare agent simulant under controlled conditions and exposure durations to evaluate the overall protective performance of the ensemble.
To understand the theories and principles of the MIST methodology, a surface-level understanding of the MIST procedures is required. Currently, there are two distinct standard methods that explain how a MIST evaluation is carried out. One method is ASTM F 2588: Standard Test
Method for Man-In-Simulant-Test (MIST) for Protective Ensembles [59], and the other is the
Military Test Operations Procedure (TOP) 10-2-022: Chemical Vapor and Aerosol System-Level
Testing of Chemical/Biological Protective Suits [60]. These two methods contain specific detail-level
77 differences in the way the test is performed and how the results are calculated, but the overall ideas
and goals are the same for each method. A discussion of how and why the standards differ is
presented in later sections (Section 4.4).
A main component in the MIST methodology is the test facility itself. Such a facility must contain an environmental chamber where the temperature, relative humidity, wind speed, and simulant concentration can be controlled and maintained to strict tolerances. Standard temperature/humidity probes and anemometers must be used to monitor the environmental conditions in real time, while specific and sensitive chromatographic or spectroscopic instruments are required to monitor the simulant concentration across the chamber area. In addition to the actual test chamber, complementary rooms are needed for donning and doffing garments as well as sample collection and
analysis. As of 2011, there are a small number of MIST facilities around the world in either military
or academic settings. The only facility found at an academic institution in the United States, which
opened in July 2008, is located at the Textile Protection and Comfort Center (TPACC) at North
Carolina State University. This MIST facility was constructed in response to the difficultly of
scheduling testing with the other facilities located at military institutions. This MIST chamber is
shown in Figure 4-1 and is approximately 300 ft2 and can accommodate up to four test subjects at one
time.
Another important element of the MIST methodology is the use of human test subjects.
Although there is an inherent variability with using human subjects, they can offer subjective
information that other testing means cannot, as well as providing the most “true-to-life” simulation of
the ensemble’s fit and usage. Prior to donning the ensemble, passive samplers are adhered to the skin
of the subjects in strategically placed locations to evaluate the protective performance of the
ensemble. A more detailed description of the samplers is given in Section 4.2.1.
78
Figure 4-1. MIST Chamber at NC State University
79 After the subjects have donned the full ensemble, they are directed to enter the test chamber where they perform a number of exercises to increase the air movement inside the garment. Since the main purpose of the MIST is to evaluate the performance of the seams, closures, and other interfaces, the exercises are designed to stretch, torque, and compress all of these areas. After a pre-determined time of exposure, the subjects exit the chamber where technicians help to doff the ensemble. The subjects are then directed to a separate room where the samplers are collected and stored for analysis.
The mass of the simulant that is detected on each of the samplers is used to generate a protection factor for that localized area. Each of the standard methods has their own Body Region Hazard
Analysis (BRHA) that has been developed to provide localized protection factors that relate to vesicant exposure and total body or systemic protection factors that predict the exposure to the nerve agents. Generally, it is necessary to evaluate one ensemble with multiple human subjects so that enough statistically significant data can be produced.
4.2 Theories and Principles of MIST Methodology
4.2.1 Methyl Salicylate as a Chemical Warfare Agent Simulant
Exposing human subjects to live CWAs to determine the chemical protective performance of an ensemble is extremely dangerous and is not considered a moral practice in today’s society as it was during World War I and II. Therefore a chemical simulant must be used in the MIST evaluations.
The selection of an appropriate chemical simulant is based on the CWAs that are of interest. As discussed in earlier sections, the main routes of exposure that must be protected are the respiratory tract and the skin. In the MIST methodology, it is assumed that an appropriate level of respiratory protection is used, so the main focus of the test is to evaluate the dermal protection that the ensembles
80 provide. The CWAs that pose the most obvious risk to the skin are vesicating agents, like sulfur
mustard (HD), because they attack the skin at the site of contact causing the formation of blisters.
However, the nerve agents with low vapor pressures, like the highly persistent VX, are also extremely
hazardous when exposed to the skin. These nerve agents can be readily absorbed through the skin
barrier and cause systemic effects throughout the nervous system. Based on this information, an
appropriate chemical simulant for the MIST must accurately simulate both sulfur mustard and VX.
Common simulants for CWAs include methyl salicylate (MeS) for sulfur mustard and dimethyl
methyl phosphonate (DMMP) for VX. It is important to realize the difference between an analog of a
CWA and a simulant. Analogs have the same basic structure as the CWA, but are still highly toxic.
Simulants normally bear no structural resemblance to the CWA, but they have similar physical
properties and are considerably less toxic [58]. Testing to compare the rates at which MeS and HD
permeate materials was conducted by the Army in 1994, and it was found that they were similar
enough to allow the use of MeS as a simulant for HD in MIST evaluations [58]. As can be seen from
Table 4-1, MeS and HD have many similar physical properties that further suggested the use of MeS
as an appropriate simulant for the vesicant. MeS has a significantly higher vapor pressure than VX so
it is likely that it not as good as simulant for the nerve agent. However, to limit the complexity of the
test method to one simulant, MeS is still used to simulate both agents. It is important to note that
since VX has a lower vapor pressure than MeS, any systemic protection factors that are calculated
using the simulant would most likely under-estimate the protection and err on the side of caution or
safety. In 2001, Riviere et al. compared the percutaneous absorption of MeS to HD using the isolated
perfused porcine skin flap (IPPSF) model and confirmed that they had similar cutaneous disposition
[61]. Another aspect that allows for MeS to be used in the MIST is its relative non-toxic nature. It is often referred to as the oil of wintergreen and is used as a flavoring in various products [58]. MeS is
81 in the salicylate family of pharmaceutical products and is used as an analgesic in sports creams and
other ointments. However, if the dose is large enough, any chemical can produce toxic or lethal
effects, so it is imperative to use caution and proper respiratory protection when handling MeS. In
fact, the 98% pure liquid form of MeS that is available over the counter contains 1400 mg of aspirin
equivalents per mL, so one teaspoon of the liquid is equivalent to taking approximately 22 adult
aspirin at one time [62]. The lowest dose of aspirin that can potentially produce toxic effects is only
150 mg/kg, and so if an average 6-year-old (23 kg) were to ingest one teaspoon of the oil of
wintergreen the total dose would be over 300 mg/kg and most likely result in lethal effects [62]. The
MIST procedures use a vapor concentration of 100 mg/m3 of MeS and the longest exposure time would be two hours so there is considerably less potential for a toxic exposure during the test duration.
Table 4-1. Structures and Physical Properties of CWAs and MeS [58]
Agent MeS HD VX
Chemical Structure
Common Name Methyl Salicylate Distilled Sulfur Mustard VX
Chemical Formula C8O3H8 C4H8Cl2S C11H26NO2PS
Molecular Weight 152 159 267 (g/mol)
Vapor Pressure 0.091 (20°C) 0.08 0.0007 (mm Hg at 25°C)
Liquid Density 1.18 1.27 1.008 (g/mL at 25°C)
Freezing Point (°C) -8.3 8-12 -39
82 4.2.2 Principles of Passive Diffusion and Passive Adsorbent Dosimeters
One of the main questions that had to be answered during the development of the MIST
methodology was how to collect and analyze any simulant vapors that entered the ensemble. Since
maintaining the integrity of the chemical protective ensemble during testing is critical to accurately
evaluating the protective performance, an ideal sampling method would be small, compact, and
completely contained inside the ensemble. Even though there are highly sophisticated spectroscopic
and chromatographic methods available, most of them are bench-level instruments that require large
amounts of power and other resources that cannot be incorporated inside an ensemble. The best
approach to analyze the amount of simulant that infiltrates the suit is through the use of air sampling
methods followed by analysis after the test is completed. Active and passive are the two main types
of sampling methods that can be used to collect any MeS vapors inside the small volume between the
subject’s skin and the garment material. Active sampling refers to the use of pumps or fans to draw
air over and through an adsorbent material to collect the compound of interest [58]. This type of
sampling is very similar to the process by which the activated carbon filters the air entering an air- purifying respirator. A specific flow rate and sampling time are used along with the mass of the chemical detected on the adsorbent to calculate the exposure concentration. The pumps or fans used in active sampling generate artificial air flows that are not normally present in the micro-environment underneath a protective garment. To better mimic the actual process that occurs when the skin is exposed to a chemical vapor, the MIST developers chose to use passive sampling. The process of passive sampling follows Fick’s laws of diffusion, whereby a chemical of interest diffuses onto an adsorbent based on the existence of a concentration gradient instead of the use of pumps or fans to draw air samples through the adsorbent. The passive samplers that are used in MIST evaluations are referred to as Passive Adsorbent Dosimeters (PADs) (Figure 4-2) and were developed by the U.S.
83 Army Natick Soldier Research, Development, and Engineering Center [58]. Requirements on the
physical dimensions and properties of the PADs as well as the manufacturing process are included in
ASTM F 2588 [59]. The PADs are constructed of a 0.025 mm thick high-density polyethylene
(HDPE) membrane that is heat sealed to the nylon/foil barrier film. An adhesive backing is applied to allow the PAD to adhere to the test subject’s skin. Approximately 40 mg of the Tenax® TA (60/80 mesh) adsorbent is sealed inside the PAD to collect the simulant vapors [59]. Tenax® TA is a porous
polymer resin based on 2,6-diphenyl-p-phenylene oxide (Figure 4-2) which has a high surface area
allowing it serve as an efficient adsorbent for volatile organic chemicals.
n
Figure 4-2. Passive Adsorbent Dosimeter and Structure of Tenax® TA
The HDPE membrane is a vital component of the sampler because its dimensions regulate the
total diffusion of the chemical into the PAD and ultimately onto the adsorbent. A diagram of the
84 process of passive diffusion through a membrane is shown in Figure 4-3. The rate at which molecules diffuse through the membrane is referred to as the sampling rate or uptake rate. The only properties of the PAD that should affect the uptake rate are the active surface area and thickness of the membrane. Other variables that affect the total uptake of a chemical into a passive sampler are the concentration gradient, time of exposure, and diffusion coefficient of the specific chemical.
Environmental conditions such as temperature, relative humidity, and wind speed can also affect the diffusion of the simulant molecules [63].
Figure 4-3. Passive Diffusion through a Permeable Membrane [64]
85 The relationship between the uptake rate and the key variables in the diffusion process is given in Equation 4-1 [63] [65]. Fick’s first law of diffusion takes into account two concentrations that form the gradient which drives the diffusion process, but with samplers the adsorbent serves as a
‘zero sink’ for the chemical which essentially makes the vapor concentration inside sampler zero.
Therefore the second concentration term is not included in Equation 4-1.
= = Equation 4-1 푚 퐷 푢 푎 u - uptake rate (cm/s)퐴퐶 푡 퐿 m - mass of analyte collected by the sampler (µg)
3 Ca - concentration of chemical in air (µg/cm ) t - time of sampling (s) D - diffusion coefficient of chemical in the membrane (cm2/s) A - surface area of membrane (cm2) L - membrane thickness (cm)
For the development of the MIST protocols, the PAD membrane was designed to limit the
uptake rate of MeS similar to the values observed for the percutaneous absorption of sulfur mustard in
skin [58]. In the 1940s two studies were conducted to measure the percutaneous absorption of sulfur
mustard into the skin. The first study by Henriques et al. evaluated the absorption when the skin was
exposed to liquid sulfur mustard [66], and the second study by Bergmann et al. investigated the
saturated vapor exposures to skin [67]. Fedele and Nelson [68] utilized the data from both studies to
86 develop a reasonable approximation for the uptake rate of sulfur mustard into skin, and their models
were presented in a report for the Army’s JSLIST development program. Due to the differences in
the composition of the skin in different body regions, the studies showed that the measured uptake
rate varies between 1-4 cm/min across the body [68]. Areas such as the perineum (area around the
genitals and anus) have higher uptake rates whereas the hands and feet have lower uptake rates.
Since it was not feasible to manufacture and keep track of specific PADs for each body region, the
MIST developers decided to follow Fedele and Nelson’s suggestion of matching the uptake rate of the
PADs to the moderate value of 2.0 cm/min for the forearm.
4.3 Differences between ASTM F 2588 and TOP 10-2-022 Methods
4.3.1 Differences between the Environmental Conditions
Although the main theories of both MIST standard methods are the same, there are various
differences between them that result from a difference in the main purpose of each standard. Just as
the NFPA 1991 and NFPA 1994 standards both give requirements for specific uses of vapor-
protective ensembles, the MIST standards are also designed to evaluate a specific type of use. The
main differences, given in Table 4-2, are in the testing conditions. The ASTM standard, which is
included in NFPA 1994 requirements, is intended to evaluate an ensemble that a first responder
would use after a CBRN attack. Since most first responders or firefighters use an SCBA, which normally has only 30-60 minutes of clean air, the exposure duration for an ASTM MIST is only 30 minutes [59]. On the contrary, military personnel mainly use air-purifying respirators which are not limited by the amount of supplied air. In the event of an attack on the battlefield, soldiers may be required to remain in their respirators for long periods of time, and therefore the TOP standard uses a
120-minute exposure time [60]. The ASTM standard provides specific conditions with tolerance
87 ranges so that each test environment is comparable across MIST trials. The TOP standard only
specifies that the facility must be capable of achieving a range for each environmental condition
because it may be beneficial to test specific combinations of temperature, humidity, wind speed, and
simulant concentrations. The typical testing conditions are actually very similar to the ASTM method.
Table 4-2. Differences in MIST Parameters for ASTM and TOP Standards [59] [60]
Test Condition or Parameter ASTM F 2588 TOP 10-2-022
27 ± 5°C 21.1 - 32.2°C Temperature (80 ± 10°F) (70 - 90°F)
% Relative Humidity 65 ± 20% 50 - 90%
Methyl Salicylate Conc. 100 ± 15 mg/m3 10 - 1,000 mg/m3 ± 5% 1.55 ± 0.65 m/s 0.89 - 4.47 m/s Wind Speed (3.5 ± 1.5 mph) (2 - 10 mph)
Number of PADs 30 23
Test Duration 30 minutes 120 minutes
4.3.2 Differences between the Exercise Protocols
Another major difference between the two standards is the exercise protocols that the subjects
are required to follow during the test. The ASTM standard incorporates exercises and movements
that a first responder may encounter such as dragging a dummy across the floor to simulate having to
88 rescue an incapacitated individual. There are also various stretches and basic calisthenics to test the integrity of the ensemble. All ten of the required exercises for the ASTM standard (Table 4-3) are conducted for one minute, resulting in a ten-minute cycle. The cycle is completed three times to achieve the 30-minute exposure [59]. The cycle of exercises in the TOP standard is also completed three times, but there are only eight exercises that must be performed for five-minute durations. As with the ASTM standard, these exercises are also designed to simulate the movements that the ensemble may experience in usage. The test subjects are required to climb a ladder, walk on a treadmill, and lie on the floor as if the soldier was taking cover.
Table 4-3. Differences in MIST Exercise Protocols for ASTM and TOP Standards [59] [60]
Station ASTM F 2588 Exercises TOP 10-2-022 Exercises
Exercise Duration 1 minute 5 minutes
Exercise 1 Dummy Drag Jumping Jacks Exercise 2 Duck Squat Sitting Rest - Right Side to Wind Exercise 3 Body Bend Walking Simulation Exercise 4 Arm Bend Sitting Rest - Left Side to Wind Exercise 5 Torso Twist Lifting Weights Exercise 6 Arm Reach Taking Cover Exercise 7 Climbing Simulation Walking Simulation Exercise 8 Crawl in Place Climbing Simulation Exercise 9 Sitting Rest - Facing Wind - Exercise 10 Sitting Rest - Back to Wind -
89 4.3.3 Differences between the PAD Placement
One of the most obvious differences between the standards is the number and placement of
the PADs on the test subject. Diagrams showing the locations of the PADs for the ASTM and TOP
methods are given in Figure 4-4 and Figure 4-5, respectively. The ASTM method requires 30 PADs that are generally split symmetrically across the body, whereas the TOP method only requires 23
PADs that are mainly placed on one side of the subject’s body.
Figure 4-4. PAD Placement Diagram for ASTM F 2588 [59]
90
Figure 4-5. PAD Placement Diagram for TOP 10-2-022 [60]
While the standards do differ in the specific placement of some of the PADs, the overall principle is the same. PADs are placed under the arm, around the ears and neck, and in the perineum to monitoring body regions that are considered vulnerable or sensitive to CWA exposure. Other
PADs are strategically placed in common interface areas such as near zippers and close to where gloves, boots, or respirators integrate into the overall garment.
91 4.4 Development and Explanation of Body Region Hazard Analysis
4.4.1 Development of Model to Predict Systemic Effects from Nerve Agent VX Exposure
A significant amount of research was conducted to develop the MIST methodologies. The first task was to identify and select MeS as an appropriate simulant for sulfur mustard. The next development was to develop a passive sampler that could be used in the micro-environment underneath a protective ensemble and determine the appropriate membrane thickness to set the sampler uptake rate close to the uptake rate of sulfur mustard into human skin. The third step was to establish all of the testing conditions, parameters, and exercise protocols that ensure each set of trials can be comparable to the next. All of the time and effort that went into these advancements would essentially be useless if there were no way to correlate the raw protection factors generated from the
PADs to specific physiological endpoints that result from exposures to both sulfur mustard and VX.
Therefore the part of the MIST methodology that is the most important to accurately evaluating chemical protective ensembles is the Body Region Hazard Analysis (BRHA). The basic idea of the
BRHA is to evaluate the protective ensemble by establishing correlations between dosage that the individual is exposed to and the agent-dependent physiological endpoints.
In 1995 Fedele and Nelson [68] developed a model to predict both the systemic effects of exposure to VX and the localized effects from sulfur mustard exposure. Their model first addressed the systemic effects that result from the cutaneous absorption after a vapor exposure. As previously discussed, nerve agents act by inhibiting the acetylcholine esterase (AChE) enzyme which induces a number physiological responses throughout the body. One response that is used to gauge the severity of the exposure to VX is the onset of nausea and vomiting. In a separate publication, Dickson and
Duncan along with Fedele and Nelson [69] describe the evolution of the model. First, they analyzed data that related the inhibition of the AChE enzyme to the probability of vomiting. Secondly, a
92 separate set of data related the intravenous dose of VX to the AChE enzyme inhibition. By
combining these sets of data, a classic S-shaped dose-response curve was developed and is given in
Figure 4-6. The data suggest that a 30% decrease in the AChE activity will produce vomiting in 50%
of the exposed population [68]. Their calculated value of 1.45 µg/kg was shown to agree with other
studies that suggested an ED50 (the required dose to achieve a specific effect in 50% of the exposed population) between 1.0 and 2.12 µg/kg for an intravenous dose of VX [68].
Figure 4-6. Dose-Response Curve for Vomiting as a Result of Intravenous dose of VX [69]
Since the effects of VX exposure are directly related to the total accumulated dosage in the body, the variability in skin absorption must be taken into account. Fedele and Nelson analyzed data that were gathered by Sim [70] on the variability of VX penetration in different body regions, and
through their model, calculated median effective doses of VX by body region (Table 4-4) [68].
93 Table 4-4. Median Effective Doses of VX by Body Region [68]
Dose Body Region Surface Area Dose Region # (mg/individual) Description Name (cm2) (µg/kg) (70kg) 1 Scalp 350 10.9 0.76 Top, back and sides of head
2 Ears 50 6.6 0.46 Outer and inner surfaces
3 Cheeks and Neck 100 6.9 0.48 Back of cheeks and sides of neck
4 Chin and Neck 200 5.1 0.36 Bottom of chin and front of neck
5 Nape 100 24.6 1.72 Back of neck
6 Abdomen 2,858 31.9 2.23 Major frontal area shoulders to groin
7 Back 2,540 37.9 2.65 Major back area shoulders to buttocks
8 Axillae 200 29.6 2.07 Armpits
9 Upper arms, medial 488 40.0 2.80 Inner upper arms
10 Upper arms, lateral 706 93.9 6.57 Outer upper arms
11 Elbowfolds 50 29.9 2.09 Crease of inner elbows
12 Elbows 50 32.1 2.25 Outer area of elbows
13 Forearms, volar 487 40.0 2.80 Flexor aspect of the forearms
14 Forearms, dorsum 706 93.9 6.57 Extensor aspect of the forearms
15 Hands, dorsum 200 41.6 2.91 Back of the hands
16 Hands, palmar 200 132.0 9.24 Palm sides of hands
17 Buttocks 953 60.9 4.26 Lower back to posterior thigh
18 Groin 300 17.4 1.22 Pelvis to coccyx, excluding perineum
19 Scrotum 200 1.6 0.11 Perineum including external genitalia
20 Thighs, anterior 2,845 93.9 6.57 Front of upper legs
21 Thighs, posterior 1,422 60.9 4.26 Back of upper legs
22 Knee 200 102.0 7.14 Kneecaps
23 Popliteal Spaces 100 29.9 2.09 Crease behind knees
24 Shins 1,897 93.9 6.57 Front of lower legs
25 Calves 948 40.0 2.80 Back of lower legs
26 Feet, dorsum 500 94.3 6.60 Top of feet
27 Feet, plantar 300 102.0 7.14 Bottom of feet
Total: 18,950
94 4.4.2 Development of Model to Predict Localized Effects from Sulfur Mustard Exposure
Instead of having to accumulate a total dosage in the body before the onset of symptoms as with nerve agents, exposure to sulfur mustard causes damaging effects at the site of contact. At the time they developed the systemic model, Fedele and Nelson did not have access to data describing the body region variability related to sulfur mustard exposure [71]. Therefore they made the assumption that if one body region is a certain times more sensitive to VX than another body region then this same body region would also be more sensitive to sulfur mustard exposure. To confirm this assumption and determine if the values for VX and sulfur mustard are similar, the absorption velocities or uptake rates that Sim observed for the volar forearm [70] were compared to the sulfur mustard exposures conducted by Henriques et al. [66] and Bergmann et al. [67]. Sim documented a range of uptake rates between 1.56 and 3.9 cm/min [68]. This range agrees well with the 2.0 cm/min value that the other studies observed. In the localized effects model, the effective dosages for each body region are calculated relative to the value for the forearm according to Equation 4-2 [68]. Using this relationship, they calculated the median effective doses for vesication by body region.
, = , , Equation 4-2 푀, 푉푋 50푗 푚퐻퐷 50푗 푚퐻퐷 50 푓표푟푒푎푟푚 � � 푀푉푋 50 푓표푟푒푎푟푚 m(HD,50j) - Median effective dose for HD vesication for body region, j
2 m(HD,50 forearm) - Median effective dose for HD vesication for volar forearm (50 mg/cm )
M(VX,50j) - Median effective dose for VX exposure for body region, j
M(VX,50 forearm) - Median effective dose for VX exposure for volar forearm (2.80 mg)
95 Table 4-5. Median Effective Doses for HD Vesication by Body Region [68]
Dose Body Region Surface Area C T m Region # (mg/individual) 0 (HD,50j) (HD,50j) Name (cm2) 3 2 (70kg) (mg.min/m ) (µg/cm ) 1 Scalp 350 0.76 271 13.6
2 Ears 50 0.46 164 8.2
3 Cheeks and Neck 100 0.48 171 8.6
4 Chin and Neck 200 0.36 129 6.4
5 Nape 100 1.72 614 30.7
6 Abdomen 2,858 2.23 796 39.8
7 Back 2,540 2.65 946 47.3
8 Axillae 200 2.07 739 37.0
9 Upper arms, medial 488 2.80 1000 50.0
10 Upper arms, lateral 706 6.57 2346 117.3
11 Elbowfolds 50 2.09 746 37.3
12 Elbows 50 2.25 804 40.2
13 Forearms, volar 487 2.80 1000 50.0
14 Forearms, dorsum 706 6.57 2346 117.3
15 Hands, dorsum 200 2.91 1039 52.0
16 Hands, palmar 200 9.24 3300 165.0
17 Buttocks 953 4.26 1521 76.1
18 Groin 300 1.22 436 21.8
19 Scrotum 200 0.11 39 2.0
20 Thighs, anterior 2,845 6.57 2346 117.3
21 Thighs, posterior 1,422 4.26 1521 76.1
22 Knee 200 7.14 2550 127.5
23 Popliteal Spaces 100 2.09 746 37.3
24 Shins 1,897 6.57 2346 117.3
25 Calves 948 2.80 1000 50.0
26 Feet, dorsum 500 6.60 2357 117.9
27 Feet, plantar 300 7.14 2550 127.5
Total: 18,950
96 In 2003, NATO released a report on a number of human chamber trials with sulfur mustard that were performed in the mid-20th century [71] [72]. These studies provided much more data on the
effects of sulfur mustard vapor exposures across different body regions and environmental conditions.
With the introduction of this data, Dickson [71] re-evaluated the localized effects model that was
developed by Fedele and Nelson. The probability (%) of observing different severities of effects
from different exposures in the various body regions is given in Table 4-6. From this data Dickson
concluded that there is a probability of experiencing at least mild effects at all of the exposure levels,
and an exposure of 50-100 mg.min/m3 resulted in severe effects in almost every body region in the
10-percentile range [71]. Whereas the previous model based the calculations on the ED50 of a liquid
VX exposure, this new data suggested that the ECt10 would be more appropriate for the sulfur mustard calculations because effects were seen at this level, and effects to only 10% of the exposed individuals would most likely be allowable [71]. Based on the data in Table 4-6, the body regions were grouped into overall regions with corresponding sensitivities. The five overall regions are: head/neck, arm/hand, perineum, torso/buttocks, and leg/foot. These regions, their observed ECt10’s and ECt50’s, and the effective dosage value that Dickson recommends for each region are presented in Table 4-7. This re-evaluation of the model for predicting the localized effects of exposure to sulfur mustard suggested that the values in the previous model were overestimating the dosage that would
3 cause severe effects. The values for C0T(HD,50j) in Table 4-5 range from approximately 40 mg.min/m
3 for the perineum to 3,300 mg.min/m for palm of the hand. Based on the new data, using the ECt10, the range would be adjusted to 25 – 100 mg.min/m3. Therefore the new model, which is incorporated
into the ASTM standard but not the TOP standard, most likely does not overestimate the dosage
required to experience severe effects. Instead, this data has allowed for the standard to err of the side
of caution when it comes to predicting the protective performance of ensembles.
97 Table 4-6. Probability (%) of Observing Effects for HD Exposure Range for the Specific Body Region [71]
Severe Effects Mild/Moderate Effects At Least Mild/Moderate Effects Body Region 25-50 50-100 100-200 200-500 25-50 50-100 100-200 200-500 25-50 50-100 100-200 200-500
mg.min/m3 mg.min/m3 mg.min/m3 mg.min/m3 mg.min/m3 mg.min/m3 mg.min/m3 mg.min/m3 mg.min/m3 mg.min/m3 mg.min/m3 mg.min/m3 Neck NA 3 22 61 NA 78 57 36 NA 80 78 97 Axillae 3 13 41 13 20 9 30 38 23 22 70 50 Penis 0 0 8 63 3 20 41 13 3 20 49 75 Scrotum 13 9 61 88 48 63 39 13 60 72 100 100 Elbowfold 5 13 61 38 25 35 27 38 30 48 89 75 Back of knee 0 2 17 38 15 13 49 25 15 15 66 63 Shoulder 3 2 41 25 13 28 43 13 15 30 84 38 Scapula 3 11 47 13 15 41 30 25 18 52 77 38 Ventral thorax 3 13 46 25 53 39 41 38 55 52 87 63 Lateral thorax 0 7 40 25 13 13 29 50 13 20 69 75 Dorsal throax 0 9 56 25 35 46 36 50 35 54 91 75 Abdomen 3 13 46 38 25 7 31 50 28 20 77 88 Groin 0 0 12 25 20 2 6 25 20 2 18 50 Buttocks 0 4 24 25 25 41 49 25 25 46 73 50 Arm 3 15 46 83 36 50 43 6 39 65 89 89 Wrist NA 15 4 17 NA 8 26 54 NA 23 30 71 Thigh 3 0 26 50 33 50 53 13 35 50 79 63 Leg 0 0 13 50 13 41 57 25 13 41 70 75
98 Table 4-7. Estimated ECt10 and ECt50 Values for Severe Effects from HD Exposure [71]
ECt ECt Recommended Body Region 10 50 Overall Body Region severe severe Value for Region
Neck 100 200 Head/neck 100 Axillae 50 150 Arm/hand 50 Elbowfold 50 100 Arm 50 200 Wrist/back of hand 50 500 Penis 200 200 Perineum 25 Scrotum 25 100 Shoulder 100 500 Torso/buttocks 100 Scapula 50 500 (excluding Ventral thorax 50 500 perineum) Lateral throax 100 500 Dorsal Thorax 100 200 Abdomen/lower 50 500 Groin 100 500 Buttocks 100 500 Back of Knee 100 500 Leg/foot 100 Thigh 100 200
Leg 100 200
With the appropriate simulant and passive sampler and with both the systemic and localized models it is possible to calculate a reasonable protection factor for a protective garment. The factors are referred to as physiological protective dosage factors (PPDF), and the specific calculations for both standard methods are presented in Appendix A.
99 CHAPTER 5: Development of Methods for Extraction and Analysis of Methyl Salicylate in
Passive Adsorbent Dosimeters
5.1 Introduction and Background
Evaluating the performance of chemical protective ensembles is the main objective of the
Man-In-Simulant-Test (MIST) method. To accomplish this goal, human subjects are fitted with
several Passive Adsorbent Dosimeters (PADs) and then exposed to a chemical warfare agent simulant
(methyl salicylate, MeS) while performing a number of different exercises in an environmental
chamber. After the exposure is complete, the PADs are collected from the subjects and analyzed to
determine the amount of MeS that entered the ensemble. The amount of MeS on each PAD is used
along with the total measured chamber concentration to generate both localized protection factors for
various body regions and a systemic protection factor for the overall total body exposure.
The MIST methodology can be separated into two different methods: the method designed to
evaluate first responder equipment which is governed by ASTM F2588, and the method intended for
military use which is stated in TOP 10-2-022. Both of these standards go into great detail about the requirements and conditions for the test chamber and facility, the placement of the PADs, the handling of the human subjects, and the overall procedure for exposing the subjects, but one area that is neglected by both standards is the method by which the PADs are analyzed. Two independent facilities could follow the specifications for the chamber and procedures for testing identically, but if one facility follows a well-defined analytical method for PAD analysis and the other does not, then it is highly likely that the results may not be in agreement. Just as in the pharmaceutical industry or with the Environmental Protection Agency, well-defined and characterized analytical methods are extremely important to ensure that each MIST facility analyzes the PADs in a consistent and reproducible manner.
100 An appropriate method would address the three distinct phases of the PAD analysis: the extraction of the Tenax® TA adsorbent from the PADs, the extraction of MeS from the adsorbent, and the analytical method used to quantify the amount of MeS in each sample. Not only is it important that the extraction and analysis methods are both accurate and precise, it is equally
important that the methods can be executed as quickly as possible. MIST trials normally involve four
to eight human subjects with thirty to forty PADs for each subject, which can total well over 250
samples to analyze for one ensemble. Long extraction or analysis times can lead to weeks or over a
month before the results from a single MIST trial are reported which is not beneficial for the
developers of new chemical protective ensembles. The objective of this research is to develop both
extraction and analysis methods to quickly and accurately analyze the amount of MeS adsorbed onto
PADs.
5.2 Experimental Methods
5.2.1 Extraction of Tenax ® from Passive Adsorbent Dosimeters
The process to extract the Tenax® from the PADs involves six simple steps which are demonstrated in Figure 5-1. After a standard MIST trial, the PADs are removed from the subject,
wrapped in aluminum foil, placed in sealed glass vials, and stored in a refrigerator at 4°C for no more
than one hour prior to extraction. Step 1 in the extraction process is to retrieve the PAD from the
glass vial and to remove the aluminum foil. The second step is to shake all of the Tenax® down to
the bottom of the PAD so that very little of the adsorbent may be lost. Step 3 involves cutting off the
top portion of the PAD so that an opening can be formed. Using a pointed pick, the polyethylene film
is pulled away from the PAD backing so that the adsorbent can be removed from the PAD (Step 4).
101
Step 1: Retrieve PAD Step 2: Shake all Tenax® down
Step 3: Cut off top of PAD Step 4: Pull film away from backing
Step 5: Vacuum Tenax® out of PAD Step 6: All Tenax® removed
Figure 5-1. Extraction Methodology of Tenax® from PAD
102 The process to remove the Tenax® from the PAD (Step 5) utilizes a 1-mL solid-phase extraction
(SPE) tube (Supelco) pre-fitted with a filter. The SPE tube is modified by installing a disposable pipet tip (0.5-20-µL ep-Tip, Eppendorf) to serve as the tip that enters the PAD. The SPE tube is connected to a vacuum source and used to remove the adsorbent from the PAD. The empty modified tube is shown in Figure 5-2a, and the tube filled with the adsorbent is shown in Figure 5-2b. The
sixth step in Figure 5-1 simply shows the PAD after all Tenax® is removed.
a) Empty SPE tube with pipet tip b) SPE tube filled with Tenax® from PAD
Figure 5-2. Modified SPE Tube Used in Tenax® Extraction from PAD
103 5.2.2 Extraction of Methyl Salicylate from Tenax ®
After the Tenax® is removed from the PAD, the second phase of the extraction method involves the removal of the MeS from the adsorbent. The SPE tubes containing the adsorbent are placed into a Supelco Visiprep DL Vacuum Manifold containing autosampler vials to collect the extract (Figure 5-3). Each SPE tube has two 600-µL aliquots of acetonitrile (ACN) (Fisher Scientific
Optima LC/MS Grade, 99.9%) injected with a repeater pipet (Eppendorf) that has a 10-mL capacity pipet tip. The ACN is allowed to flow through the adsorbent to extract the MeS. A low vacuum (-2 in Hg) is turned on for one minute to drain the last drops of extract out of the tubes, and the vials are capped and stored for later analysis.
Figure 5-3. Vacuum Manifold Used to Extract Methyl Salicylate from Tenax®
104 Some of the solvent volume (1,200 µL) used to extract the MeS remains in the disposable liner that is placed in the vacuum manifold or is adsorbed by the Tenax®. The manifold has a 12- vial/sample capacity so one vial in each manifold is weighed before and after extraction to determine the final weight of solvent. This value is converted to volume of solvent using the density of ACN
(0.787 g/mL) [73].
5.2.3 High Performance Liquid Chromatography (HPLC) Analysis Method
The PADs were analyzed on an Agilent 1260 Series HPLC containing a Binary Pump SL
(Model Number G1312B), a standard autosampler (Model Number G1329B), a thermal column compartment (Model Number G1613A) and a diode-array detector (Model Number G4212B). The injector syringe was rinsed in ACN (Fisher Scientific Optima LC/MS Grade, 99.9%) to prevent carryover, and the injection amount was 4 µL. For highly concentrated specimens, the injection amount was decreased to 0.5 µL in order to avoid overloading the detector. An Agilent Poroshell 120
EC-C18 column (3.0 mm x 100 mm, 2.7µm particle size) heated to 45°C was used with a flow rate of
0.5 mL/min. A 40/60 H2O/ACN isocratic method was used with a total analysis time of 4.0 minutes.
The diode-array detector was set to monitor at 310 nm.
5.2.4 Development of Calibration Curve for Methyl Salicylate
To develop a calibration curve for MeS on the HPLC, a 30,000 ng/µL stock solution was
prepared by weighing out 3.000 g of MeS into a 100-mL volumetric flask and filling to volume with
ACN (Fisher Scientific Optima LC/MS Grade, 99.9%). A 300 ng/µL stock solution was made by
diluting 1 mL of the 30,000 ng/µL solution in a 100-mL volumetric flask. Likewise, a 30 ng/µL stock
105 solution was made by diluting 10 mL of the 300 ng/µL solution in a 100-mL volumetric flask, and finally, a 3 ng/µL stock solution was prepared by diluting 10 mL of the 30 ng/µL solution in a 100- mL volumetric flask. From these less concentrated stock solutions, the calibration standards given in
Table 5-1 were made. Solutions #1 – #13 were made by diluting the appropriate amount of the 3 ng/µL stock solution into 10-mL volumetric flasks. The 30 ng/µL stock solution was used to make
Solutions #14 – #16.
Table 5-1. Methyl Salicylate Calibration Solutions for HPLC
ng/PAD ng/PAD Conc. Solution # 4-µL Injection 0.5-µL Injection (ng/uL) (1 mL of Solvent) (1 mL of Solvent)
1 0.003 3 24
2 0.009 9 72
3 0.015 15 120
4 0.021 21 168
5 0.03 30 240
6 0.09 90 720
7 0.15 150 1,200
8 0.21 210 1,680
9 0.3 300 2,400
10 0.9 900 7,200
11 1.5 1,500 12,000
12 2.1 2,100 16,800
13 3.0 3,000 24,000
14 6.0 6,000 48,000
15 9.0 9,000 72,000
16 15 15,000 120,000
106 The detection range for the analytical method must be quite large because PADs tested in a very protective ensemble may adsorb less than 100 ng while PADs used to measure the chamber concentration may adsorb over 100,000 ng. One method to achieve such a large range of detection is shown by the injection amounts in Table 5-1. Samples that were exposed on the test subject can be assumed to be on the lower end of the range, so a 4-µL injection is used on the HPLC to introduce more sample onto the column and increase the sensitivity. On the contrary, PADs that are fully exposed in the chamber can be assumed to have MeS amounts on the higher end of the range, so a
0.5-µL injection is used on the HPLC so as to not overload the detector. The same calibration solutions can be used for both injection amounts, but if the overall calibration is based on the 4-µL injections, it is necessary to multiply the results by a factor of eight when running samples with the
0.5-µL injection. To generate the calibration curve for the method, all standards were analyzed on the
HPLC in triplicate with a 4-µL injection.
5.2.5 Calculation of Method Detection Limit and Limit of Quantitation
Because a chemical protective ensemble is designed to limit the amount of the simulant that enters the suit, a high performing ensemble will have very low amounts of MeS detected on the subject PADs. Therefore the method detection limit (MDL) or limit of detection (LOD) and limit of quantitation (LOQ) are extremely important for PAD analysis methods. There are two distinct sets of
MDL and LOQ values when discussing the analysis of PADs; the first set of values refers specifically to the detection limits for the analytical method while the second set of values characterizes the detection limits for analyzing PADs. The MDL and LOQ values for the analytical method do not take into account the PADs and are only based on standard solutions of MeS. The PAD MDL and
107 LOQ values should be higher than those for the instrument and method because of impurities or
interferences that can be in the PADs.
The methods used to calculate the MDL and LOQ values for this research were based on a
report by the Wisconsin Department of Natural Resources Laboratory Certification Program [74]. To
determine the detection limits for the analytical method, a sample of the 0.003 ng/µL (3 ng/PAD)
standard was analyzed a total of nine times. The MDL was calculated by multiplying the sample
standard deviation of the nine replicates by 2.896, which is the Student’s T-value that corresponds to
eight degrees of freedom at the 99% confidence level. The LOQ was calculated by multiplying the
sample standard deviation by a factor of ten.
The MDL and LOQ values were determined for the PAD analysis method by extracting
thirty-six blank PADs in three separate vacuum manifolds. The MDL was calculated by multiplying
the sample standard deviation of the HPLC results by 2.434, which is the Student’s T-value that corresponds to thirty-five degrees of freedom at the 99% confidence level. The MDL was also calculated for the twelve PADs in each manifold using the sample standard deviation and the
Student’s T-value that corresponds to eleven degrees of freedom at the 99% confidence level (2.681).
5.2.6 Determination of Variability in Solvent Volume Calculation
To determine the variability in the amount of solvent that actually ends up in the vials after the extraction, thirty-two blank PADs were extracted in three separate manifolds and the weights of vials were measured before and after the extraction process. All vials were weighed with the caps so that none of the solvent would evaporate during the weighing process.
108 5.2.7 Determination of Extraction Efficiency for Common Solvents
An important aspect of the extraction method is the choice of an appropriate solvent that
quickly and efficiently extracts the MeS from the Tenax®. To determine the extraction efficiency of
various solvents, four sets of twelve PADs were exposed in the full-scale MIST chamber at North
Carolina State University for sixty minutes at an average MeS concentration of 107.31 mg/m3. The
PADs were extracted using two 600-µL aliquots of acetonitrile (ACN) (Fisher Scientific Optima
LC/MS Grade, 99.9%), isopropanol (IPA) (Fisher Scientific HPLC grade), methanol (MeOH) (Fisher
Scientific GC Resolv), and n-hexane (Fisher Scientific GC Resolv). Four vacuum manifolds were used in the extraction, and the twelve PADs for each solvent were extracted in the same vacuum manifold. All vials were weighed before and after the extraction to determine the volume of solvent in the vial. After the PADs were extracted, the vials containing the extract were replaced with empty vials and a second extraction was conducted to determine if any MeS remained in the Tenax® after the first extraction.
5.2.8 Determination of Extraction Efficiency at Various Spiked Amounts
To determine the extraction efficiency across the range of MeS amounts expected on the various PADs, sets of PADs were spiked with the stock solutions using a syringe from an autosampler on a gas chromatograph. For this experiment, a set of PADs refers to six PADs. Four sets of PADs were spiked with 1 µL of the 30 ng/µL, 300 ng/µL, 3,000 ng/µL, and 30,000 ng/µL stock solutions. The PADs were wrapped in aluminum foil, placed in sealed glass vials, and stored in a refrigerator for one hour prior to extraction. All vials were weighed before and after the extraction to determine the volume of solvent in the vial. After the PADs were extracted, the vials containing
109 the extract were replaced with empty vials and a second extraction was conducted to determine if any
MeS remained in the Tenax® after the first extraction.
5.3 Results and Discussion
5.3.1 Extraction of Tenax® from Passive Adsorbent Dosimeter
The most important aspects of an acceptable process to extract the Tenax® from the PADs are the speed and efficiency of the method. The extraction process presented in Section 5.2.2 incorporates both of these aspects of performance. By using four technicians working with six vacuum manifolds, all of the PADs for four MIST subjects can be processed and readied for MeS extraction in only 1 – 2 hours. As can be seen in the photo for Step 6 in Figure 5-1, nearly all of the adsorbent particles are removed from the PAD, and due to the vacuum line and filter, the particles are held tightly inside the SPE tube. Other possible extraction methods may involve multiple transfers of the adsorbent such as moving from the PAD to a tube and them from a tube into a vial. Each additional transfer introduces opportunity for loss of sample. Therefore, the NCSU PAD extraction method has been developed to incorporate only one sample transfer, that of going from the PAD to the SPE tube.
5.3.2 Development of Analytical Method and Calibration Curve for Methyl Salicylate
One of the first steps in developing an analytical method for a specific compound is to determine which type of instrument is the most appropriate. Since MeS is relatively volatile, gas chromatography (GC) can and has been used for the PAD analysis. Analysis by GC coupled with either a thermal desorption unit or a mass spectrometer were initially investigated as potential
110 analytical instruments for MeS analysis. While each individual method has its own strengths, one of the major drawbacks to using a GC is that a considerable amount of the analysis time per sample is dedicated to simply heating or cooling the oven, which leads to 10 – 15 minutes to analyze a single sample. As stated previously, one of the major goals of this research is to develop the quickest possible analysis method. With this objective in mind, the long analysis times using a GC method are unacceptable. One solution to this problem is to develop an isocratic method using liquid chromatography because there is no down time wasted on heating or cooling an oven. The sample analysis time for the HPLC method that has been developed is only 3 – 4 minutes per sample. The short run time on the HPLC has allowed for the analysis of PADs from four MIST subjects to take only 10 – 12 hours as opposed to 36 – 40 hours for a GC method.
A short analysis time was not the only factor that contributed to the selection of an HPLC method. Since the amounts of MeS adsorbed onto PADs used in MIST trials can cover a significantly wide range, the analytical method must also be capable of detecting MeS across multiple orders of magnitude. The chromatogram in Figure 5-4 shows the detection of MeS on a ppb level, which corresponds to approximately 9 ng/PAD. This chromatogram was generated using a 4-µL injection.
Methyl Salicylate Retention Time: 1.88 min Conc. 0.009 ng/µL ~ 9 ng/PAD with 4-µL injection
Figure 5-4. Chromatogram of Low-Level Methyl Salicylate Standard at 310 nm
111 On the other hand, the chromatogram in Figure 5-5 shows the detection of MeS nearly four orders of magnitude higher (90,000 ng/PAD) using a 0.5-µL injection. The flexibility of use different injection amounts on the HPLC allows for the analysis of a wide range of concentrations using the same basic method. The 4-µL injection increases the sensitivity of the method because more sample is introduced onto the column. Likewise, a highly concentrated sample such as a fully exposed chamber PAD can be analyzed with the 0.5-µL injection without overloading the column and potentially leading to carryover in the following samples.
Methyl Salicylate Retention Time: 1.88 min Approximate Conc. 10.4 ng/µL ~ 90,000 ng/PAD with 0.5-µL Injection
Figure 5-5. Chromatogram of High-Level Methyl Salicylate PAD Extract at 310 nm
The average of three replicates for each calibration solution described in Section 5.2.4 was used to develop both the low and high range calibration curves given in Figure 5-6 and Figure 5-7.
The corresponding calibration equations are also given by Equation 5-1 and Equation 5-2. The low range has a correlation coefficient of 0.999996 while the high range has a coefficient of 0.999950.
112 200 y = 57.761x + 0.0776 180 R² = 1 160
140
120
100 AreaCounts 80
60
40
20
0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Methyl Salicylate Concentration (ng/µL)
Figure 5-6. Low-Range Methyl Salicylate Calibration Curve on HPLC at 310 nm (Equivalent to 3-3,000 ng/PAD with a 4-µL injection)
900
800 y = 55.882x + 3.226 R² = 1 700
600
500
AreaCounts 400
300
200
100
0 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 Methyl Salicylate Concentration (ng/µL)
Figure 5-7. High-Range Methyl Salicylate Calibration Curve on HPLC at 310 nm (Equivalent to 12,000-120,000 ng/PAD with a 0.5-µL injection)
113 Low Range Calibration Equation
= 57.761( ( )) + 0.078 Equation 5-1
퐴푟푒푎 푀푒푆 퐶표푛푐푒푛푡푟푎푡푖표푛 푛푔⁄µ퐿
High Range Calibration Equation
= 55.882( ( )) + 3.226 Equation 5-2
퐴푟푒푎 푀푒푆 퐶표푛푐푒푛푡푟푎푡푖표푛 푛푔⁄µ퐿
5.3.3 Calculation of Limit of Detection and Limit of Quantification
While it has been shown that the HPLC method does provide a rapid analysis of samples and
can detect MeS in the wide range needed for PAD analysis, one aspect that may be even more
important is the detection limit. If a chemical protective ensemble performs as designed, it should
allow very little MeS to enter the garment and be adsorbed onto the PADs. Therefore, the ideal analytical method will be capable of detecting MeS at very low levels.
In the case of PAD analysis for MIST samples, detection limits must be calculated for the analytical method using standard solutions, but these limits must also be calculated for the analysis of actual PADs. Therefore, two sets of detection limits have been calculated for the NCSU PAD analysis method. Using a method defined by the Wisconsin Department of Natural Resources
Laboratory Certification Program [74], the MDL and LOQ for the HPLC method has been
determined. A 0.003 ng/µL sample was chosen to determine the detection limits because it was the
lowest calibration standard that was prepared. The sample was analyzed a total of nine times and the
results are given in Table 5-2. Based on a 4-µL injection volume and 1 mL of extraction solvent, the
114 MDL for the analytical method is 1.3 ng/PAD (0.001 ng/µL) and the LOQ is 7.8 ng/PAD (0.008
ng/µL). These detection limits show that the HPLC and analytical method are capable of accurately
distinguishing a liquid sample of MeS at very low levels.
Table 5-2. Method Detection Limit and Limit of Quantitation for Analytical Method
Method Detection Limit Limit of Quantitation Average Standard Sample ID Area Deviation Area ng/µL ng/PAD Area ng/µL ng/PAD
0.003 ng/µL 0.358 0.053 0.153 0.001 1.3 0.529 0.008 7.8
Since clean calibration standards are the ideal scenario to detect extremely low levels of MeS, it is reasonable to assume that the analysis method will have be slightly less sensitive when analyzing the extract from the PADs. The MDL and LOQ values for the PAD analysis were calculated by analyzing three sets of twelve blank PADs. Each set was extracted in a separate manifold, and the results for each manifold, along with the total overall results, are given in Table 5-3. It can be seen from these data that the samples that have been extracted through blank PADs produce slightly higher
MDL and LOQ values. Based on a 4-µL injection and 1 mL of solvent, the overall MDL for all thirty-six PADs was 7.3 ng/PAD (0.007 ng/µL) and the LOQ was 34.1 ng/PAD (0.034 ng/µL).
Comparing these detection limits to those of the analytical method show that the process of passing the solvent through the PAD can affect the analysis results. This effect may be due to contaminants or interferences from the PAD that generate a baseline with more noise. More noise in the baseline makes it more difficult to distinguish an extremely small sample, and therefore the detection limits
115 increase. Although the MDL and LOQ values are higher for the PAD analysis method, it is important
to note that they are still in a reasonably low range. If it is desirable to extend the limits even lower, it
may be possible to use only one 600-µL aliquot of the solvent to extract the MeS from the PADs. It is reasonable to assume that this change could decrease the LOQ value for the PAD analysis method closer to 20 ng/PAD, but a significant amount of work would need to be conducted to ensure that the smaller volume would efficiently extract all MeS from the PADs.
Table 5-3. Method Detection Limit and Limit of Quantitation for PAD Analysis
Method Detection Limit Limit of Quantitation Average Standard Sample ID Area Deviation Area ng/µL ng/PAD Area ng/µL ng/PAD
Manifold #1 0.644 0.142 0.381 0.005 5.3 1.422 0.023 23.3
Manifold #2 0.664 0.217 0.582 0.009 8.7 2.171 0.036 36.2
Manifold #3 0.652 0.257 0.688 0.011 10.6 2.566 0.043 43.1
Total 0.654 0.205 0.498 0.007 7.3 2.048 0.034 34.1
5.3.4 Determination of Variability in Solvent Volume Calculation
Since some of the injected solvent is trapped in the Tenax® or in the disposable liner during the extraction process, the variability in the final solvent volume was investigated. Three sets of twelve blank PADs were put through the extraction process in three separate vacuum manifolds.
Each vial was weighed before and after extraction to determine the weight and ultimately, the volume
116 of ACN that ended up in the vial. The average solvent volume for all twelve PADs in each manifold
with the 95% confidence intervals are shown in Figure 5-8, and a basic statistical breakdown of the
data is given in Table 5-4. On average, only 86-88% of the 1.200 mL that was injected in each
sample was collected in the vials. However, the method is relatively consistent considering that the
average solvent volume for all thirty-six vials in the three manifolds was 1.054 ± 0.4% (based on the
95% confidence interval). Using this average and confidence interval, a PAD sample found to have
0.3 ng/µL of MeS would correlate to an amount of 316 ± 1 ng of MeS. Even if the highest or lowest volume out of the thirty-six samples was used to calculate the total mass of MeS on the PAD, the range would only be 309 – 323 ng. This tight range shows that the appropriate solvent volume can be calculated by only weighing one vial from each manifold as opposed to every vial that is used.
1.200
1.150
1.100
1.050
1.000
0.950 Volume of(mL) Solvent Volume 1.058 1.063 1.036 0.900
0.850
0.800 Manifold 1 Manifold 2 Manifold 3
Figure 5-8. Average Volume of Acetonitrile Collected After Extraction for Each Manifold
117 Table 5-4. Basic Statistical Analysis of Solvent Volumes
Average Volume Standard 95% Confidence Maximum Minimum Sample ID (mL) Deviation Interval Value Value
Manifold #1 1.040 0.007 0.004 1.054 1.031
Manifold #2 1.058 0.010 0.006 1.075 1.041
Manifold #3 1.063 0.007 0.005 1.077 1.052
Total 1.054 0.013 0.004 1.077 1.031
5.3.5 Extraction Efficiency for Common Solvents
Another critical part of characterizing the extraction process is determining the efficiency at which the solvent extracts MeS from the Tenax®. Choosing the appropriate or highest efficiency solvent adds another layer of confidence to the extraction methodology. Therefore, an investigation into the extraction efficiency of four common solvents was conducted by exposing PADs in the full- scale MIST chamber, extracting a separate set of PADs with each solvent, and conducting a second extraction to determine the amount of MeS left in the adsorbent after the first extraction. The average results of both extractions with each solvent are shown in Figure 5-9. The first observation that can be made is that after completing two extractions, all four solvents removed similar amounts of MeS from the PADs, approximately 74,000 – 80,000 ng. It is also clear from the data that ACN is the most efficient solvent for this extraction method. The calculated extraction efficiency for the first extraction with ACN was 99.0 ± 0.2%. The same value for IPA was 66.9 ± 0.9%, for MeOH was
86.4 ± 0.7%, and for n-hexane was 82.3 ± 1.9%. Based on the fact that none of the efficiencies for
118 the other solvents were remotely close to the extraction efficiency of ACN with one extraction, it has been selected as the most appropriate solvent for the MeS extraction process.
1st Extract 2nd Extract 90,000
80,000 820 10,896 70,000 13,575 24,844 60,000
50,000
40,000 78,032 69,452 30,000 62,559 50,128 Amount of Methyl Salicylate (ng) Salicylate Methyl of Amount 20,000
10,000
0 Acetonitrile Isopropanol Methanol Hexane
Figure 5-9. Extraction Efficiency of Common Solvents
5.3.6 Determination of Extraction Efficiency at Various Spiked Amounts
Since ACN has been shown to have the highest efficiency of the tested solvents, it was important to determine the extraction efficiency across the range of expected MeS amounts. The previous section discussed PADs that were fully exposed in the MIST chamber for an hour at full concentration, but the majority of MIST samples are only exposed to low concentrations that enter the ensemble. To determine the extraction efficiency of ACN across the range of MeS amounts, sets of
119 PADs were spiked with 30 ng, 300 ng, 3,000 ng, and 30,000 ng. These PADs were then processed
through two extractions as discussed in the previous section. The average extraction efficiencies for
each spike level are shown in Figure 5-10. From the data, it can clearly be seen that ACN is fully
capable of extracting MeS from the Tenax® at efficiencies between 98% and 99% across the full
range of MeS amounts.
1st Extract 2nd Extract
100 2.1 1.2 1.6 0.6
80
60
97.9 98.8 98.4 99.4 40 % Extraction Efficiency Extraction %
20
0 30 300 3,000 30,000 Amount of Methyl Salicylate Spiked onto PAD (ng)
Figure 5-10. Extraction Efficiency across Range of Expected Methyl Salicylate Amounts
5.4 Conclusions
In conclusion, the results prove that the NCSU PAD extraction and analysis methods have met or exceeded the project objectives. An adsorbent extraction method has been developed that can
120 quickly and efficiently remove the Tenax® from the PADs with only one transfer step that limits the potential loss of any adsorbent particles during the process. With only four technicians, the method has be shown to be capable of extracting all PADs for a four subject MIST in as little as 1-2 hours.
The extraction of MeS from the adsorbent using ACN has been shown to be over 98% efficient across the expected range of MeS amounts, and ACN was shown to have a much higher extraction capability than IPA, MeOH, or n-hexane. After the Tenax® has been extracted from the
PADs this technique has shown to be capable of removing the MeS and having the samples ready for
HPLC analysis in a matter of minutes as opposed to letting the samples sit in vials for hours to complete the extraction. Using the vacuum manifold consistently yields an average solvent volume of 1.054 mL ± 0.4%. This consistency allows for only one vial to be weighed from each manifold as opposed to weighing every vial that is used in a MIST trial which further speeds up the analytical process.
The HPLC method was shown to provide both fast analysis and high sensitivity for analyzing
MeS. The MDL and LOQ values for the analytical method using calibration standards were 1.3 ng/PAD and 7.8 ng/PAD respectively. As expected, the MDL and LOQ values for the PAD analysis method were higher at 7.3 ng/PAD and 34.1 ng/PAD, respectively, most likely due to contaminants or interferences that were extracted from the PADs.
Overall the NCSU PAD extraction and analysis method provides extremely fast and efficient methods to confidently and accurately analyze the samples generated in a MIST trial. Utilizing every aspect of the presented research, NCSU’s MIST facility is capable of conducting, analyzing, and reporting a four subject ASTM test in as little as three to four days.
121 CHAPTER 6: Development of Bench-Scale Methyl Salicylate Exposure Chamber
Excerpts from the following section were part of a conference report for the AATCC 2010
International Conference and Exhibition in Atlanta, Georgia in May 2010.
6.1 Introduction and Background
The newly constructed Man-In-Simulant-Test (MIST) Facility at North Carolina State
University (NCSU) has supplied the chemical protection community with an alternative location to conduct full-scale MIST trials with human subjects, but more importantly, it has provided a means to conduct much needed research on the various components of the test itself. Although the MIST chamber is an invaluable tool when it comes to the evaluation of full ensembles on human subjects or large-scale testing of the Passive Adsorbent Dosimeters (PADs) at uniform conditions, a bench-scale approach offers the flexibility to quickly test small lots of PADs or other materials at multiple conditions. The MIST chamber requires an extended period of time to achieve a steady state condition due to the large volume of the chamber and also requires a number of technicians to ensure that the test runs smoothly. A bench-scale chamber allows for a single technician to quickly expose small lots of samples under various simulant concentrations.
Similar research on the development of a bench-scale exposure chamber was conducted at
Battelle Memorial Institute in 2006 [75]. The focus of this research was to compare three different types of passive sampling devices according to their diffusive uptake rates of methyl salicylate (MeS), an extensively used simulant for chemical warfare agents. One of the examined devices, the Natick
Sampler, is the PAD that is specified in the ASTM F 2588 standard for MIST testing [59]. Battelle’s research in the bench-scale chamber yielded a 12.7 cm3/min calculated uptake rate for the Natick
122 Sampler PAD, which is slightly larger than the 10 ± 2 cm3/min uptake rate specified in the ASTM
standard [75] [59].
The main goals for this research are to design a bench-scale MIST chamber, to validate that the chamber can repeatedly maintain specific simulant concentrations for long periods of time, and to compare PAD exposures between the bench-scale chamber and the full-scale MIST facility.
6.2 Experimental Methods
6.2.1 Design and Construction of Bench-Scale Chamber
The basic dimensions of the bench-scale MIST chamber were developed so that a human arm
or manikin arm form could easily fit inside. The concept drawings for the chamber are given in
Figure 6-1, with the drawing on the left showing the end cap that enables the insertion of an arm form
and the drawing on the right showing the end cap used in normal PAD exposures. The chamber is
constructed of a one-inch thick acrylic cylinder that is two feet in length with an outer diameter of ten
inches resulting in a the total chamber volume of approximately twenty-three liters.
Figure 6-1. SolidWorks® Visualization of Bench-Scale Chamber Design
123 The end caps are machined out of a two inch thick acetal copolymer (McMaster-Carr) cylinder that fits tightly into the chamber body. Each end cap contains four equally spaced vapor entrance or exit ports that are fitted with ¼” Swagelok® fittings. Each of the end caps are tightly secured to the chamber body through the use of four adjustable draw latches (McMaster-Carr) with a ten-inch rubber gasket placed between the end cap and cylinder body. The cylinder body is suspended between two PVC pipes that are attached to a Plexiglas® foundation. The fully constructed bench-scale MIST chamber is shown in Figure 6-2.
Figure 6-2. Bench-Scale MIST Chamber
As depicted in Figure 6-2, the methyl salicylate flow enters the chamber on the right and exits the chamber on the left. The entrance end cap has a quick-access door built in so that samples can be introduced into the chamber with minimal disruption to the chamber conditions. This access door also contains a small fan intended to provide air movement for mixing of the simulant (Figure 6-3).
124
Figure 6-3. Chamber End Cap with Quick-Access Door Closed (left) and Open (right)
6.2.2 Methyl Salicylate Vapor Generation System
One of the most critical components of the bench-scale MIST chamber is the generation of the MeS vapor. It is imperative that the vapor generation be controllable and capable of maintaining the concentration in the chamber for extended periods of time. The design of the bench-scale vapor generation system was heavily based on the design of the same system for the full-scale MIST facility at NCSU. As shown in Figure 6-4, a 100-mL round-bottom three-neck flask is partially filled with liquid MeS (99%, Acros Organics) and heated in a heating mantle to a desired temperature. One of
125 the necks of the flask holds the thermocouple that monitors the liquid MeS temperature, while the other two necks are for the air entrance and exit. All of the lines that carry the vaporized MeS are ¼” stainless steel, heated with a heating tape, and insulated with a heat resistant meta-aramid fabric. A vacuum is used to pull air into the chamber and generate an overall negative pressure, thus limiting the opportunity for MeS to leak out of the chamber. The total flow through the chamber can be varied in the range of 1 – 10 L/min. The flow required to pass through the MeS boiler is much lower than the overall flow through the chamber (between 0.1 and 1.0 L/min), and is controlled with a needle valve and monitored with the mass flow meter shown in Figure 6-4. A basic schematic for the flow through the chamber is given in Figure 6-5.
Figure 6-4. Methyl Salicylate Vapor Generation System for Bench-Scale MIST Chamber
126
Figure 6-5. Flow Schematic for Bench-Scale MIST Chamber
6.2.3 Methyl Salicylate Concentration Measurement
As shown in Figure 6-5, the flow exiting the chamber is directed to an FT-IR system where real-time concentration measurements are taken. The CIC Photonics gas cell FT-IR (Figure 6-6) provides approximately twenty individual measurements per minute and is calibrated by the manufacturer to accurately measure MeS concentrations in the range of 1 – 50 ppm (~6 – 300 mg/m3). The chamber also has a second optional system for measuring the MeS concentration. This air sampling system (Figure 6-7) draws a low flow (~60 mL/min) through tubes packed with Tenax®
TA (60/80 mesh) to collect the MeS exiting the chamber. After a defined sampling duration, the adsorbent can be removed from the tubes, the MeS can be extracted from the adsorbent, and the total mass of MeS collected is used to calculate the chamber concentration.
127
Figure 6-6. CIC Photonics Gas Cell FT-IR Measurement System
Figure 6-7. Air Sampling System for Bench-Scale MIST Chamber
128 6.2.4 Investigation into the Repeatability of Chamber Conditions
After the construction of the chamber, it was necessary to determine the specific set points for
each of the parameters that affect the simulant concentration. For this research, the total flow through
the chamber, the heating tape temperature, and the MeS boiler temperature were all set to constant values. The chamber temperature and relative humidity were monitored and generally stayed close to the ambient conditions in the laboratory. Currently there is no mechanism to control temperature, but desiccant tubes can be placed in the flow path to achieve a humidity level that is lower than the ambient conditions. The set points for each of the parameters are given in Table 6-1.
To develop a calibration curve for the simulant concentration, the flow through the MeS
boiler was varied from 0.1 – 0.8 L/min at 0.1 L/min increments. The chamber concentration was
allowed to stabilize for sixty minutes. This set of experiments was conducted twice to determine the
repeatability of the chamber.
Table 6-1. Bench-Scale Parameter Set Points
Chamber Parameter Set Point Value Chamber Parameter Set Point Value
MeS Boiler Temperature 50°C Chamber Temperature 22-24°C Heating Tape Temperature 50°C Chamber Relative Humidity 30-50% Total Chamber Flow 7 L/min Chamber Fan 1.6 m/s
129 6.2.5 PAD Exposure Methodology
Three PAD exposure trials were conducted in the bench-scale chamber to determine the
repeatability of the method. Ten PADs were exposed for 30 minutes at average concentrations of
16.31ppm (101.52 mg/m3), 16.30 ppm (101.46 mg/m3), and 16.16 ppm (100.58 mg/m3) for trials 1, 2,
and 3, respectively.
PAD exposures were conducted to compare the total amounts of MeS adsorbed in both the
bench-scale chamber and the full-scale MIST chamber. In both the chambers, twelve Natick Sampler
PADs (M&C Specialties Co.) from lot P3268 were exposed at 100 mg/m3 for sixty minutes. The
PADs were removed from each chamber and extracted immediately.
6.2.6 PAD Extraction Methodology
An extraction method developed at North Carolina State University was employed to recover the MeS from the PADs. After exposure, the adsorbent was removed from each PAD through the use of a vacuum and a modified empty 1-mL SPE tube (Supelco) with a filter in the tip. The SPE tubes containing the adsorbent were then placed into a Supelco Visiprep DL Vacuum Manifold with autosampler vials to collect the extract. Each SPE tube had two 600-µL aliquots of acetonitrile
(Fisher Scientific Optima LC/MS Grade, 99.9%) injected with a repeater pipet that had a 10-mL capacity pipet tip. The acetonitrile was allowed to flow through the adsorbent to extract the MeS, and then a low vacuum (-2 in Hg) was turned on to drain the last drops out of the tubes, and the vials were capped and stored for later analysis.
130 6.2.7 High Performance Liquid Chromatography (HPLC) Analysis Method
The PADs were analyzed on an Agilent 1260 Series HPLC containing a Binary Pump SL
(Model Number G1312B), a standard autosampler (Model Number G1329B), a thermal column
compartment (Model Number G1613A) and a diode-array detector (Model Number G4212B). The
injector syringe was rinsed in acetonitrile (Fisher Scientific Optima LC/MS Grade, 99.9%) to prevent
carryover, and the injection amount was 4 µL. For highly concentrated specimens, the injection
amount was decreased to 0.5 µL in order to avoid overloading the detector. An Agilent Poroshell 120
EC-C18 column (3.0 mm x 100 mm, 2.7µm particle size) heated to 45°C was used with a flow rate of
0.5 mL/min. A 40/60 H2O/ACN isocratic method was used with a total analysis time of 4.0 minutes.
The diode-array detector was set to monitor at 310 nm.
6.3 Results and Discussion
6.3.1 Consistency and Repeatability of Chamber Conditions
The most critical part of the bench-scale chamber development was the ability to control and
maintain a desired MeS concentration for a long period of time. In order to calculate the appropriate
flow rate through the MeS boiler to achieve the simulant concentration of 16 ppm (100 mg/m3) required by the ASTM standard [59], it was necessary to first develop a relationship between the flow rate and the overall chamber concentration. Using the set points given in Table 6-1 and varying the
MeS boiler flow rate, the chamber concentration profiles in Figure 6-8 were generated. Each time the flow rate was changed, the chamber was allowed to equilibrate for an hour. The first observation that can be made from this data is that 10 – 15 minutes after the flow rate was changed, the concentration reached a steady state condition and maintained that level for the remainder of the test duration.
131 26
24
22
20
18
16 0.80 L/min 0.70 L/min 14 0.60 L/min 0.50 L/min 12 0.40 L/min 0.30 L/min 10 0.20 L/min MeS Concentration (ppm) MeS Concentration 8 0.10 L/min
6
4
2
0 0 10 20 30 40 50 60 Test Duration (minutes)
Figure 6-8. Bench-Scale MIST Chamber Concentration Profiles
Another observation from the chamber profiles is that the relationship between the MeS boiler flow rate and the chamber concentration does not appear to be linear. The chamber concentration increases at a higher rate than the increase in the flow rate. This relationship is further shown in Figure 6-9, which gives the average steady state concentration of two replicate runs at each
flow rate (the error bars indicate the 95% confidence interval). The relationship fits very well with a
2nd degree polynomial, and the tight interval on most of error bars show that the chamber can repeatedly be filled to the same concentration when the same parameters are used.
132 26 24 y = 14.919x2 + 14.937x + 1.0795 R² = 0.9999 22 20 18 16 14 12 10 8
Chamber Concentration (ppm) Chamber Concentration 6 4 2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Boiler Flow Rate (L/min)
Figure 6-9. Relationship between MeS Boiler Flow Rate and Chamber Concentration
= Equation 6-1
퐶1푉1̇ 퐶2푉̇2 - Steady state MeS concentration (ppm) of chamber (from FT-IR)
ퟏ 푪 - Total chamber flow rate (L/min)
푽̇ ퟏ - Steady state MeS concentration (ppm) of boiler flow ퟐ 푪 - Boiler flow rate (L/min)
푽̇ ퟐ
133 Assuming that the chamber has reached a steady state condition, the concentration of MeS in the boiler flow stream can be calculated by a simple molar dilution equation (Equation 6-1). Using this equation, the average steady state chamber concentrations, and the corresponding flow rates, the concentrations for the boiler flow streams were calculated and are shown in Table 6-2. The overall trend, with the exception of the data point at 0.1 L/min, is an increase in the concentration of MeS in the boiler flow stream as the flow rate through the boiler increases. These data suggest that the chamber concentration increases according to the 2nd degree polynomial in Figure 6-9 because the air
passing over the vaporized MeS in the boiler picks up more MeS as the flow rate increases.
Table 6-2. Calculated Concentration of MeS in Boiler Flow Stream
Trial/Rep #1 Trial/Rep #2 MeS Boiler Calculated MeS Calculated MeS Average MeS Chamber Conc. Chamber Conc. Flow Rate Boiler Conc. Boiler Conc. Boiler Conc. (ppm) (ppm) (L/min) (ppm) (ppm) (ppm) 0.1 3.0 211.9 2.5 175.5 193.7 0.2 4.7 164.1 4.5 158.4 161.2 0.3 6.6 153.3 7.0 164.0 158.7 0.4 9.6 168.5 9.5 167.0 167.8 0.5 12.3 171.5 12.4 173.5 172.5 0.6 15.4 180.2 15.3 178.1 179.1 0.7 18.7 186.9 18.9 189.0 187.9
0.8 22.7 198.5 22.5 197.3 197.9
134 6.3.2 Repeatability of PAD Exposures in Bench-Scale Chamber
The repeatability of the bench-scale chamber was determined by exposing PADs in three separate trials. A basic statistical analysis of the trials is given in Table 6-3. It can be seen that the amount of MeS adsorbed on the PADs in each trial was very consistent with an average across all trials of 60,100 ± 3.5% (interval based on 95% confidence interval). These data along with the relationship shown in the previous section prove that the bench-scale chamber can be filled to a desired concentration and the results from separate trials can be compared with a high degree of confidence.
Table 6-3. Basic Statistical Analysis of Bench-Scale Chamber Trials
Statistical Value Trial 1 Trial 2 Trial3 All Trials
Average Amount (ng) 60,265 60,104 59,930 60,100
Standard Deviation 6,021 4,280 6,850 5,614
95% Confidence 4,307 3,062 4,900 2,096
% Coefficient of 10.0 7.1 11.4 9.3 Variation
6.3.3 Comparison of Bench-Scale and Full-Scale MIST Chamber PAD Exposures
PAD exposures were conducted in both the bench-scale and full-scale MIST chambers to determine the agreement between the two different approaches. The target exposure concentration
135 for the trials was 16 ppm (100 mg/m3) to reflect the ASTM standard concentration. The average MeS
concentration profiles for both chambers are given in Figure 6-10 with the tolerance limits
represented by the horizontal dashed purple lines and the test duration marked by the dashed vertical lines. The average value for the bench-scale chamber was 15.58 ppm (97.08 mg/m3) and 16.96 ppm
(105.54 mg/m3) for the full-scale MIST chamber. The spikes in the bench-scale chamber profile occur due to opening and closing the access door to insert or remove the samples. The concentration in both chambers remained within the specified tolerance limits during the duration of the tests.
Full-Scale MIST Chamber Bench-Scale MIST Chamber 25
20
15
10 Methyl Salicylate Concentration (ppm) Concentration Salicylate Methyl 5
0 -10 0 10 20 30 40 50 60 70 Time of Exposure (minutes)
Figure 6-10. Methyl Salicylate Concentration Profiles for Bench-Scale and Full-Scale Chambers
136 During the bench-scale trial, the flow through the MeS boiler had to be increased from 0.62
L/min to 0.67-0.68 L/min to maintain the concentration. Due to the small volume of the chamber it is very likely that the twelve PADs removed enough MeS from the air within the chamber to significantly affect the measured concentration. Since the volume of the full-scale MIST chamber is approximately four orders of magnitude larger than that of the bench-scale chamber, the PADs have virtually no effect on the full-scale chamber concentration.
The average amounts of MeS detected on the exposed PADs are shown in Table 6-4 along with the standard deviations and 95% confidence intervals. The most important observation from the data is that the PADs exposed in the bench-scale chamber adsorbed significantly more MeS than those exposed in the full-scale MIST chamber, and they also had a slightly higher degree of variability shown by the percent coefficient of variation. This increase in the amount of adsorbed
MeS is most likely a result of an environmental difference between the two chambers.
Table 6-4. Comparison of Basic Statistical Values for the Bench-Scale and Full-Scale Exposures
Statistical Value Bench-Scale MIST Full-Scale MIST
Average Amount (ng) 100,359 73,223
Standard Deviation 9,396 5,185
95% Confidence 5,970 2,189
% Coefficient of Variation 9.4 7.1
137 As previously stated, the MeS concentration in the bench-scale chamber was approximately
97 mg/m3 while that of the full-scale MIST chamber was 105 mg/m3. A difference of only 8 mg/m3 should not be enough to affect the adsorption on the PADs in the magnitude that was observed.
Likewise, the temperature and relative humidity during the tests were very similar, 23°C and 45% in the bench-scale and 25°C and 50% for the full-scale. With all of the other conditions being similar, the difference between the air flow dynamics in the two chambers may be responsible for the increased amount of MeS adsorbed by the PADs. In an unpublished report for the U.S. Army Natick
Research, Development, and Engineering Center, Rivin et al. explain that the uptake rate of passive samplers, like those used in the MIST, can decrease at low tangential face velocities because of a resistance to diffusion in the boundary layer [76]. Based on this information, it is logical to assume that there is a considerably higher air flow over the face of the PADs in the bench-scale chamber as opposed to the full-scale MIST chamber. Future research can be conducted in the bench-scale chamber to determine if the velocity of the fan does affect the uptake of the PADs. Until these data are generated, the best approach is to only compare results between trials conducted in the same chamber scale.
6.4 Conclusions
The main purpose of this research was to develop a bench-scale MIST chamber to aide in preliminary research on PADs or real-time MeS sensors. The bench-scale MIST chamber developed at NCSU has shown to be repeatable and to maintain a desired concentration for an extended period of time. The chamber can be set to a specific concentration according to the 2nd degree polynomial
relationship that was observed. Also, the results from separate trials showed that the bench-scale chamber produces a consistent testing environment. The comparison between the bench-scale and
138 full-scale exposure suggested that the air flow over the PADs in the bench-scale method increases the uptake rate and therefore adsorption of the PADs. Even with this drawback, the bench-scale chamber can still serve as a starting point for investigations into the characteristics of the PADs, the effectiveness of real-time sensors, or the uptake rates of other materials.
Further advances in the environmental controls of temperature and relative humidity conditions will allow for more testing possibilities. Also incorporation of flow controllers instead of only flow meters would allow for tighter control of the chamber concentration.
139 CHAPTER 7: Factors Influencing the Uptake Rate of Passive Adsorbent Dosimeters
Excerpts from the following section are from the article titled “Factors Influencing the
Uptake Rate of Passive Adsorbent Dosimeters Used in the Man-In-Simulant-Test” by B. Ormond et al. which was accepted for inclusion into the Journal of ASTM International and scheduled to be published in May 2012.
7.1 Introduction and Background
In the mid-1990s, the Man-In-Simulant-Test (MIST) was developed by a task group of the
U.S. Army Chemical and Biological Defense Command (CBDCOM) to aid in the assessment of chemical protective ensembles [58]. During the development of the test multiple questions had to be answered such as:
• What chemical could safely be used to simulate chemical warfare agents that are locally
acting vesicants (e.g. sulfur mustard or lewisite) and also systemically acting nerve agents
(e.g. Sarin or VX)?
• What is the most appropriate method to collect any of the simulant vapors that may enter the
garment while it is being tested?
• At what uptake rate does the human skin absorb the warfare agents and the simulant?
A technical assessment of the MIST program was conducted and published in 1997 [58], and
the answers to many of the task group’s questions can be found in this evaluation. The most
appropriate simulant for sulfur mustard (HD) was found to be methyl salicylate (MeS), commonly
known as the oil of wintergreen [58]. The work conducted to determine that MeS could adequately
serve as a simulant for HD mainly focused on the rates at which both chemicals permeate fabrics.
140 According to the assessment [58], MeS was shown to permeate fabrics about 30% slower than the rate of HD. However, due to the impermeable nature of the fabrics being tested, the amount of the vapor that enters the suit by permeating the material will be much less than the amount that breaches the interfaces in the suit (e.g. zippers and seams). Riviere et al. have also investigated the use of MeS as a simulant for HD by analyzing its percutaneous absorption through the use of an isolated perfuse porcine skin flap, and found that it was indeed an adequate simulant [61]. The technical assessment determined that while MeS was a good simulant for HD, its physical properties were too different from VX or Sarin to say that it would simulate the nerve agents as well as the vesicants [58].
However, MeS was chosen to represent both classes of chemicals in order to simplify the test instead of developing different tests for the two different simulants.
One of the biggest issues facing the development of the test was the method of vapor collection. Small packages of an adsorbent material that can be adhered to the test subject, referred to as Passive Adsorbent Dosimeters (PADs), work through the process of passive diffusion. Therefore no artificial airflows are generated inside the protective garment during testing as would be the case with an active sampling system that pulls air into the sampler with the use of a pump or vacuum. The passive diffusion process allows for a sampling method that is more consistent with the process of percutaneous absorption. The main parameter that needs to be defined to ensure that the PAD is consistent with the percutaneous absorption of HD is the uptake rate or sampling velocity.
Two studies conducted in the early 1940s [66] [67] looked at the absorption rates of HD into human skin. The first study exposed skin to liquid HD while the second focused on vapor exposures.
In a report for the JSLIST program in 1995, Fedele and Nelson [68] used both of these studies to develop a reasonable approximation for the uptake rate of HD into human skin. Their study makes it clear that because the thickness and sensitivity of human skin varies across the body and since the
141 uptake rate can increase with increasing skin temperatures, an ideal scenario would be to have
multiple PADs with different uptake rates for each of these different body regions [68]. The technical assessment states that the observed variation in the skin uptake rates can range between 1.0 – 4.0 cm/min [58]. However, due to the complexity of developing and dealing with multiple samplers,
Fedele and Nelson recommended that a single passive sampler should be used with an uptake rate of
2.0 cm/min, which corresponds closely with the observed uptake rate for the forearm [68].
The PAD design that is currently used, referred to as the Natick Sampler, was developed by the U.S. Army Natick Soldier Research, Development, and Engineering Center. The PAD, as shown in Figure 7-1, is constructed of a permeable high density polyethylene (HDPE) film that is heat sealed against an aluminum foil backing and also has an adhesive that allows the PAD to be attached to the test subject. The pocket inside the PAD contains approximately 40 mg of a polymer resin based on
2,6-diphenyl-p-phenylene oxide that adsorbs the simulant molecules as they diffuse through the
HDPE membrane [59]. Detailed specifications for each component of the PAD are stated in ASTM F
2588 [59].
Figure 7-1. Passive Adsorbent Dosimeter Used in MIST Protocol. Dimensions of Active Sampling Surface Area are 2.5 cm x 1.8 cm (ASTM F 2588).
142 In 2010, an ASTM subcommittee was tasked with responding to issues surrounding the calculated uptake rates of the PADs. The main issue being that different MIST facilities were reporting a wide range in the calculated uptake rates of the PADs. As previously stated, the PADs were designed to have an uptake rate of 2.0 cm/min and laboratories were reporting values as low as
1.0 cm/min and as high as 3.5 cm/min. Many manufactured products inherently have some degree of variability from lot to lot, and the PADs used for MIST testing are no different. In fact, the testing community had previously voiced concern with the manufacturing processes because the PADs varied significantly in the amount of adsorbent that they contained. In an attempt to resolve these inconsistencies, the supplier of the Natick Sampler transferred the manufacturing of the PADs from
ITW Devcon1 in Massachusetts to M&C Specialties Co.2 in Pennsylvania in the later part of 2010.
The “New” PADs (used for this study) appear to be more uniform and contain the appropriate amount
of adsorbent, but the other physical properties have not been investigated. With knowledge of these
previous issues, the quality control for PAD manufacturing was a logical starting point to determine
the source of the observed inconsistencies in the uptake rates.
When discussing the uptake rate of the PADs there are two different values that must be
considered. For clarity, the first value can be referred to as the film uptake rate (cm/min). This
parameter refers to the diffusion across the HDPE membrane and is dependent upon the thickness
(standard value of 0.025 mm [59]) and other properties of the film. The film uptake rate most closely
relates to the sampling velocity or flux referred to in the percutaneous absorption process described
by Fedele and Nelson [68]. Therefore to achieve the suggested value of 2.0 cm/min, the thickness of
the film and diffusivity of MeS in the film are the main properties to consider. Whereas the film
uptake rate is mainly dependent on these two properties, the second value, the PAD uptake rate, is
1 ITW Devcon 30 Endicott Street, Danvers, MA 01923 2 M&C Specialties Co., 90 James Way, Southampton, PA 18966 (800)-370-1575
143 dependent on both the film properties and the active sampling surface area of the PAD. The PAD
uptake rate is expressed in units of cm3/min and is a product of the active sampling surface area of the
PAD and the film uptake rate. The PAD uptake rate is the value that the current version of ASTM F
2588 simply refers to as the uptake rate. According to the standard, PADs with an active sampling surface area of 4.3 cm2 should have a PAD uptake rate of 10 ± 2 cm3/min [59]. Specifying the uptake
rate in terms of cm/min describes the diffusion process in a one-dimensional direction and only places
tolerances on the properties of the film. However by incorporating the surface area of the PAD and setting criteria for the uptake rate in terms of cm3/min, the three-dimensional diffusion process is
better described. In other words, since the membrane thickness is held constant, diffusion occurs at
the same rate (2.0 cm/min) over the whole surface of the PAD, but a larger surface area will allow
more molecules to diffuse through the membrane and be collected by the adsorbent. The opposite
would be true for a smaller active sampling surface area allowing fewer molecules to diffuse. If the
surface areas of the PADs are not consistent and held to strict tolerances, the calculation of the film
uptake rates can be incorrect.
The need for an understanding of the factors that affect the PAD uptake rate, manufacturing or
otherwise, is best shown by its importance to the MIST methodology. Testing according to the
current ASTM F 2588 standard requires that the uptake rate of each lot of PADs be measured prior to
conducting MIST trials. The accepted practice is to expose at least four PADs in the MIST chamber
for 30 ± 5 minutes to calibrate the lot of PADs used in the specific trial [59]. The average uptake rate
(cm/min) and average surface area (cm2) for each lot of PADs being used in an individual MIST experiment are then applied to all of the PADs that are attached to the test subjects. The standard states that a set of PADs from the lot must be exposed and the uptake rates are to be calculated using
Equation 7-1 [59].
144 = Equation 7-1 푚 푢 퐴퐶푡 u - Uptake rate (cm/min)
m - Mass of methyl salicylate adsorbed on PAD (ng) A - Active sampling surface area of PAD (cm2)
3 Ct - Chamber exposure dosage (mg.min/m )
( ) = Equation 7-2 퐶푡표푢푡푠푖푑푒 푃푟표푡푒푐푡푖표푛 퐹푎푐푡표푟 푃퐹 퐶푡푖푛푠푖푑푒 PF - Raw protection factor for specific PAD location (unit less)
3 Ctoutside - Exposure dosage for chamber (mg.min/m )
3 Ctinside - Measured exposure dosage for each PAD location (mg.min/m )
An average uptake rate is calculated for the lot of PADs, and then this average rate is used in
Equation 7-1 along with the detected amount of MeS from each test PAD location to calculate an exposure dosage or Ctinside for each area under the garment. Then the Ctinside for each PAD location is put into Equation 7-2 along with the exposure dosage for the chamber (Ctoutside), which for an ASTM protocol would be 100 mg/m3 for 30 minutes or a Ctoutside of 3,000 mg.min/m3. It is important to note that the Ctoutside according to the ASTM standard is not measured with PADs; on the contrary, it is an average of the real-time chamber concentration measurements typically taken with FT-IR instrumentation [59]. The ratio in Equation 7-2 provides the raw protection factors (PF) for each PAD location. These protection factors are then converted to localized and systemic
145 physiological protective dosage factors (PPDF) by multiplying them by body site specific weighting factors that reflect the varying susceptibilities of the different body regions to the chemical agents.
The purpose of this research is to answer questions that have been raised about the consistency of the PADs, and to determine which factors affect the uptake rate that is observed. In this study, the physical dimensions of the PADs were measured and examined to determine the variation that exists in the surface areas. At this time the variation in the film thickness was not measured due to a lack of instrumentation with proper sensitivity. Also, PADs were exposed to various concentrations for different lengths of time to determine the effect of the exposure dosage on the uptake rate. Another suspected source of inconsistency that was investigated was the amount of time the PADs were allowed to sit after exposure and the temperature in which they were stored.
7.2 Experimental Methods
7.2.1 Measurement of the PAD Physical Dimensions
To determine the variability in the physical dimensions, twenty PADs from the same manufacturer-sealed package had the lengths and widths measured with calipers (Mitutoyo Model
No. CD-6” CS). The measurements were taken from the edge where the film was heat-sealed (shown in Figure 7-1) and not the overall outer dimensions of the PAD.
7.2.2 Results from All PAD Exposures
In many of the experiments, a set of multiple PADs were exposed at each of the different conditions. In most cases, four PADs have been used to make a set of PADs, and any results
146 mentioned in the discussion are the average of the four PADs in the set. Using multiple PADs at each
condition provides replicate measurements for statistical importance.
7.2.3 Effect of Challenge Concentration and Exposure Time on PAD Uptake Rate
To determine how the calculated uptake rate of the PADs was affected by the challenge
concentration and exposure time, a set of experiments was carried out in the full-scale MIST chamber
at North Carolina State University. All of the PADs used in this study were the Natick Sampler and
came from same manufacturing lot (P3257). The MIST chamber was held at the conditions stated in
ASTM F 2588 (27°C, 60% relative humidity, and a nominal wind speed of 1.65 m/s). The simulant
concentration was set at five different levels for the testing, and the average concentrations measured
by a CIC Photonics Gas-Cell FT-IR were 15.2, 24.5, 55.7, 71.2, and 97.5 mg/m3. The PADs were
exposed to each of the concentrations for eleven different exposure times: 1, 5, 10, 15, 30, 45, 60, 75,
90, 105, and 120 minutes. Each combination of concentration and time produced an exposure dosage,
Ct, value allowing for comparison of a short duration-high concentration exposure to long duration-
low concentration exposure. In other words, a 30-minute exposure at 100 mg/m3 and a 100-minute exposure at 30 mg/m3 both produce a Ct value of 3,000 mg.min/m3. A set of four PADs was exposed
for each of the eleven times of exposure to ensure an accurate representation of the adsorption
process. Each time the chamber was set to a new concentration all 44 PADs for that experiment were
placed in the chamber simultaneously. Each set of four PADs was adhered to individual aluminum
plates for each exposure time. After exposed for the desired amount of time, the plate was removed
and the PADs were extracted immediately.
Due to concerns that the PADs may saturate under normal testing conditions (30 minutes at
100 mg/m3), a set of four PADs was exposed overnight in the MIST chamber at 100 mg/m3. This
147 length of exposure was over forty times the longest exposure required in the standard. After the
PADs were exposed they were extracted immediately and analyzed.
A second experiment was set up to expose the PADs to concentrations that were below the
detection limit of the FT-IR. The lowest range that was measurable by the FT-IR was approximately
10-15 mg/m3. A VICI Metronics Dynacalibrator (Model 340) with a Type D diffusion vial (NIST
traceable calibration and a diffusion path cut to one inch) was used to generate MeS vapor at
concentrations in the range of 1-5 mg/m3. The MeS flow out of the generator was directed to a glass chamber where the PADs were placed. After the gas generator was allowed to stabilize, the PADs were placed in the glass chamber and exposed for 120 minutes. Four thermal desorption tubes packed with 200 mg of clean adsorbent (60/80 mesh) were placed at two tees in the flow path. A low vacuum drew 20 mL/min through each of the tubes during the 120-minute exposure. Following the exposure the PADs and tubes were extracted immediately and prepared for analysis.
7.2.4 Effect of Post-Exposure Time on PAD Uptake Rate
A separate experiment was carried out to investigate the effect of post-exposure storage time on the total adsorption of MeS. For this experiment, a set of PADs consisted of eight total PADs, four with the adhesive backings and four with the adhesive removed. The adhesive was removed manually from the back of the PAD by peeling it off, and isopropanol wipes were used to remove any residue that remained. Seven sets of PADs were placed in the MIST chamber simultaneously and exposed for 30 minutes at 100 mg/m3. After exposure, one set of PADs was extracted immediately, while the other six sets were wrapped in aluminum foil, placed in individual sealed glass vials, and stored in a refrigerator at 4°C for six different time periods. The stored PADs were extracted after 30 minutes, one hour, two hours, four hours, one day, and three days.
148 7.2.5 Effect of Storage Temperature on PAD Uptake Rate
The effect of the storage temperature on the total amount of adsorbed MeS was examined
through two separate experiments. In the first experiment, three sets of four PADs were exposed for
30 minutes at approximately 94 mg/m3. One set was extracted immediately, one set was stored under dry ice (below -30°C) for one day, and the final set was stored in the refrigerator (1-4°C) for one day.
Again, all PADs that were stored were wrapped in aluminum foil and placed in individual sealed glass vials. In the second experiment, three sets of six PADs were exposed for 120 minutes at an average concentration of 108 mg/m3. One set was extracted immediately, one set was stored in the
refrigerator (1-4°C) for one day, and one set was stored in a freezer (-15 to -20°C) for one day.
7.2.6 Lot-to-Lot Variability in PAD Dimensions and Uptake Rate
A follow-up investigation was conducted to determine the lot-to-lot variability in both the physical dimensions of the PADs and the calculated uptake rates. The dimensions of 48 PADs were measured as described in Section 7.2.1from lot P3268 (a different lot than the PADs used in the rest of this study). To evaluate the uptake rate of the second lot of PADs, twelve PADs were exposed in the full-scale MIST chamber for 60 minutes at an average concentration of 107.3 mg/m3.
7.2.7 PAD Extraction Methodology
An extraction method developed at North Carolina State University was employed to recover
the MeS from the PADs. After exposure, the adsorbent was removed from each PAD through the use
of a vacuum and a modified empty 1-mL SPE tube (Supelco) with a filter in the tip. The SPE tubes
containing the adsorbent were then placed into a Supelco Visiprep DL Vacuum Manifold with
149 autosampler vials to collect the extract. Each SPE tube had two 600-µL aliquots of acetonitrile
(ACN) (Fisher Scientific Optima LC/MS Grade, 99.9%) injected with a repeater pipet that had a 10- mL capacity pipet tip. The ACN was allowed to flow through the adsorbent to extract the MeS.
A low vacuum (-2 in Hg) was turned on to drain the last drops out of the tubes, and the vials were capped and stored for later analysis.
7.2.8 Supplemental Extraction Methods for Desorption Tubes and Adhesive Film
The desorption tubes used to measure the MeS concentration for the 1-5 mg/m3 PAD exposure had the adsorbent removed and placed in a glass vial with 2 mL of acetonitrile. The specimens were allowed to sit for 30 minutes at room temperature. These extract specimens were then filtered into vials using 3-mL Luerlok syringes with 0.2 µm pore size Whatman filters attached.
The adhesive film specimens from the overnight exposure were extracted in the same manner, but they were allowed to sit overnight before being filtered.
7.2.9 High Performance Liquid Chromatography (HPLC) Analysis Method
The PADs were analyzed on an Agilent 1200 Series HPLC containing a Binary Pump SL
(Model Number G1312B), a standard autosampler (Model Number G1329B), and a diode-array detector SL (Model Number G1315C). The injector syringe was rinsed in acetonitrile (Fisher
Scientific Optima LC/MS Grade, 99.9%) to prevent carryover, and the injection amount was 4 µL.
For highly concentrated specimens, the injection amount was decreased to 0.5 µL in order to avoid overloading the detector. An Agilent Poroshell 120 EC-C18 column (3.0 mm x 100 mm, 2.7µm particle size) heated to 45°C was used with a flow rate of 0.5 mL/min. A 40/60 H2O/ACN isocratic
150 method was used with a total analysis time of 4.0 minutes. The diode-array detector was set to 205 nm. (Note: This wavelength is different than in other sections because after this research was conducted, the wavelength was changed to 310 nm to avoid contaminants in the PADs)
7.3 Results and Discussion
7.3.1 Variation in Physical Properties of the PADs
The first attempt to characterize some of the inter-laboratory variation was to investigate how well the active sampling surface areas of the PADs agreed with the tolerances stated in ASTM F
2588. The ASTM standard only specifies tolerance criteria for the surface area and not the individual length and width values. The active surface of a PAD is supposed to be 2.5 cm x 1.8 cm with a surface area of 4.3 ± 0.6 cm2 [59]. After measuring the dimensions of twenty PADs from one sealed pouch, it was observed that the average length was 2.38 cm, the average width was 1.78 cm, and the average surface area was 4.25 cm2. The percent deviation from the standard length and width with their corresponding surface areas are shown in Figure 7-2 and Figure 7-3 respectively. The dotted lines in these two graphs show the tolerance values for the surface area. For the most part, all but three or four PADs out of the twenty that were measured fall within the surface area tolerances.
However, it is clear from the figures that the lengths of the PADs are much more variable than the widths. This may be an issue that the manufacturer could improve upon to produce a more consistent product. As it stands from this analysis, the average surface area for the lot of PADs that would be used in the uptake rate calculation is only 1.2% smaller than the standard area that is specified. Even though the average of the measured surface areas fall within the tolerance limits, there are a few of the data points that are as much as 20% smaller and 13% larger than the standard value.
151 6.00
) 5.00 2
4.00
3.00
2.00 Active Sampling Surface Area (cm Area Surface Sampling Active 1.00
0.00 -20.0 -15.0 -10.0 -5.0 0.0 5.0 10.0 15.0 20.0 % Deviation from Standard Width
Figure 7-2. Calculated Percent Deviation from the Standard Length of 2.5 cm for 20 PADs
6.00
) 5.00 2
4.00
3.00
2.00
Active Sampling Surface Area (cm Area Surface Sampling Active 1.00
0.00 -20.0 -15.0 -10.0 -5.0 0.0 5.0 10.0 15.0 20.0 % Deviation from Standard Length
Figure 7-3. Calculated Percent Deviation from the Standard Width of 1.8 cm for 20 PADs
152 To quantify the lot-to-lot variability in the physical dimensions, a second set of PADs from a different lot were examined. The PADs discussed in the previous section were part of lot P3257 manufactured in November 2010 while the second lot of PADs (P3268) was manufactured in October
2011. The average surface areas for each lot and the 95% confidence intervals are given in Figure
7-4, and it can be seen that while the average surface areas for both lots are within the specified tolerances in ASTM F2588, there is a significant difference between the two lots. Lot P3257 has an average surface area of 4.25 cm2 with a 95% confidence interval of 0.18 cm2. On the other hand, lot
P3268 has a higher average surface area (4.75 cm2), but it has a much tighter 95% confidence interval of 0.04 cm2. These data suggest that there can be a significant difference between PADs from different lots, and it may be necessary to adjust the tolerance criteria in the standard.
5.50 ) 2
4.90
4.30
3.70 Active Sampling Surface Area (cm Area Surface Sampling Active
3.10 PAD Lot #P3257 PAD Lot #P3268
Figure 7-4. Variability in the PAD Surface Area between Lots (Error Bars Indicate the 95% Confidence Interval and Dotted Lines Indicate the Surface Area Tolerance According to ASTM F 2588)
153 7.3.2 Uptake Rate Variation with Challenge Concentration and Exposure Time
With over 200 PADs exposed and analyzed in the first set of experiments, a massive amount of data has been collected. There are numerous ways to organize the data in an attempt to pick out patterns and see which factors affect the PADs the most. It is possible to graph the total MeS adsorbed versus either the chamber concentration or the exposure time, but a significant amount of information is not gathered in regards to the uptake rate. A graph of the total MeS adsorbed versus the exposure dosage is the best way to see if the uptake rate stays constant over all of the conditions because the Ct takes into account both the time of exposure and the challenge concentration.
180000 y = 14.292x + 291.01 R² = 0.9958 160000
140000
120000
100000
80000
60000
Total MeS Adsorbed (ng) 40000
20000
0 0 2000 4000 6000 8000 10000 12000
Exposure Ct (mg.min/m3)
Figure 7-5. Total Methyl Salicylate Uptake (ng) Versus Exposure Ct (mg.min/m3). Each Data Point is the Average of Four PADs. Slope Corresponds to a PAD Uptake Rate of 14.3 cm3/min and a Film Uptake Rate of 3.4 cm/min
154 In Equation 7-1, the PAD uptake rate (with the area included) is essentially the slope of mass versus Ct linear relationship. The graph given in Figure 7-5 clearly shows that this rate is constant in the range of exposures used in this experiment. This relationship also confirms that a short duration- high concentration exposure is very similar to a long duration-low concentration exposure. Taking all points into consideration, the slope of the curve correlates to a PAD uptake rate of 14.3 cm3/min and a film uptake rate of 3.4 cm/min, if the previously measured average active sampling surface area of
4.25 cm2 is used. Both of these rates are higher than the values required in the standard or suggested
in the literature (10 cm3/min and 2 cm/min). Since the measured surface areas for this particular lot of PADs are within the standard tolerances, it should be safe to conclude that the higher uptake rates are more likely due to a property of the film as opposed to the surface area of the PAD. Therefore for the remainder of the discussion, the uptake rates will be expressed in terms of the film uptake rate in cm/min.
One important factor that affects the uptake rate can be observed if it is plotted against the time of exposure. Figure 7-6 shows that there is at least a 10 to 15-minute equilibration time before the uptake rate reaches a steady state. This realization is very important because if one laboratory only exposes PADs for 5-10 minutes to calculate the lot’s average uptake rate and another laboratory exposes PADs for more than 30 minutes, the two calculated values may not agree. The first laboratory will generate uptake rates around 1-2 cm/min while the second laboratory will calculate the steady state uptake rates around 3-4 cm/min. This relationship between the exposure time and uptake rate could contribute significantly to the inconsistencies between laboratories.
The values in Table 7-1 provide the averages, standard deviations, and coefficients of variation for all of the PADs that were exposed. The top portion of the table includes every data point while the bottom portion only includes the steady state data for PADs exposed for more than 30
155 minutes. The total average uptake rate for all of the exposures was 3.3 cm/min with a standard deviation of 0.7 cm/min. The coefficient of variation being around 20% shows that many of the data points were not at an equilibrium steady state. Taking only the PADs that were exposed for a longer period of time, the average uptake rate increases to 3.5 cm/min with a standard deviation of 0.4 cm/min, and the coefficient of variation was cut almost in half from 20% to 11%. The best recommendation based on this relationship is that PADs should be exposed for at least 30 minutes in order to achieve a steady state. Therefore the 1, 5, 10, and 15-minute exposures have been disregarded for most of the remaining discussion.
15 mg/m3 25 mg/m3 56 mg/m3 71 mg/m3 98 mg/m3 5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5 Uptake Rate (cm/min) 1.0
0.5
0.0 0 20 40 60 80 100 120 Time of Exposure (min)
Figure 7-6. Average Uptake Rate (cm/min) Versus the Time of Exposure (Minutes). Average Steady State Uptake Rate Approximately 3.5 cm/min.
156 The follow-up investigation involving the lot-to-lot variability in uptake rate showed that the
PADs from the new lot (P3268) had an average film uptake rate of 2.6 cm/min as opposed to the 3.5 cm/min for lot P3257. Since these PADs were exposed under similar conditions it is logical to conclude that slight variations in the properties of the HPDE film can affect the uptake of MeS. This finding further reinforces the notion that each lot of PADs must thoroughly be characterized prior to use in a MIST trial.
Another question that has been raised about the PADs that can be answered by Figures 4 and
5 is whether or not unprotected/fully exposed PADs would saturate under normal test conditions. An
ASTM standard test results in an exposure dosage of 3,000 mg.min/m3 and the military TOP 10-2-
022 procedure, which is a 120-minute test at the same concentration, results in a 12,000 mg.min/m3 exposure dosage. Neither of the figures shows any indication of saturation during the test. The curve in Figure 4 stays linear all the way to the 12,000 mg.min/m3 mark, and a decrease in the uptake rate would be expected if saturation were present in Figure 7-6. The PADs that were exposed overnight adsorbed an average of 775,000 ng of MeS. Using the equation of the line in Figure 4 to extrapolate the saturation point, the PADs most likely reached saturation between eight and nine hours of exposure or a Ct of approximately 54,000 mg.min/m3. Without further investigation into the prolonged exposures, a more accurate exposure dosage for saturation cannot be stated.
A third observation that can be made from Figure 7-6 and also from the data in Table 7-1 is that the uptake rate slightly increases as the concentration decreases. The values go from approximately 3.4 cm/min at the highest concentration to around 4.0 cm/min at the lowest concentration. It is important to note that the standard deviation and coefficient of variation for each concentration level are very similar. This observed consistency shows that there is an increasing trend and that the difference is not simply part of the overall variability. This trend is a critical
157 observation because even though the MIST chamber is maintained at 100 mg/m3 during testing, the microenvironment beneath the protective ensemble should be nowhere near as concentrated. The chamber concentrations used in this experiment only spanned one order of magnitude and the uptake rate increased 0.6 cm/min. Simulant concentrations inside a protective ensemble may be closer to two or more orders of magnitude lower than the chamber concentration.
Table 7-1. Statistical Values for the Variation in the Calculated Uptake Rates. Data for “All Exposed PADs” are Averaged from 44 Total PADs at Each Concentration. Data for “PADs Exposed for 30+ Minutes” are Averaged from 28 total PADs at Each Concentration.
Conc. 15.2 24.5 55.7 71.2 97.5 Total (mg/m3)
Average 3.8 3.4 3.1 3.0 3.1 3.3 (cm/min) All Exposed PADs Std Dev 0.5 0.7 0.7 0.6 0.6 0.7
%CV 14.6 20.2 21.8 21.7 20.7 21.6
Average 4.0 3.7 3.3 3.2 3.4 3.5 (cm/min) PADs Exposed for 30+ min Std Dev 0.3 0.3 0.3 0.3 0.3 0.4
%CV 6.5 8.7 7.6 8.1 8.9 11.0
If the uptake rate continues to increase at lower concentrations, then the protection factor calculations used in the MIST analysis may be incorrect because those calculations assume that the
158 uptake rate is constant at both the high chamber concentrations and the low concentrations underneath
the protective ensemble.
To estimate the concentrations expected under a chemical protective garment, one can consider Equation 7-2. According to the NFPA 1994 standard [50], a passing protection factor for a
Class 2 ensemble should be 360. Since Equation 7-2 is a ratio of the Ctoutside to the Ctinside, and the
time of exposure is the same for both cases, the time cancels out leaving only the concentrations
outside and inside the protective suit as part of the equation. Using the 360 protection factor and the
outside concentration of 100 mg/m3, the potential internal concentration for a marginally protective
garment would be 0.28 mg/m3, which is almost two orders of magnitude lower than the lowest
concentration previously discussed. Although it would have been ideal to expose the PADs to
concentrations on this level or lower, time and instrumentation constraints only allowed for a lowest
achievable concentration of 4-5 mg/m3 using the gas generator and small-scale chamber. For a 120- minute exposure at 4.8 mg/m3, the average calculated uptake rate was 4.1 cm/min with a standard deviation of 0.3 cm/min. Without further testing at even lower concentrations, it is impossible to determine how much more the uptake rate may increase.
7.3.3 Effect of Post-Exposure Storage Time and Temperature on the PAD Uptake Rate
An easily overlooked factor that could be a major source of the variation between laboratories
is in the handling of the PADs after exposure. The standard requires that the PADs be wrapped in
aluminum foil, placed in sealed glass vials, and stored in the refrigerator around 4°C [59]. Aluminum
foil is used so that there is a solid surface attached to the adhesive during storage. The adhesive is
most likely a polymeric substance that can allow the MeS to adsorb and then desorb while being
stored. Placing the aluminum foil on the adhesive was an attempt by the standard committee to
159 minimize mass transfer out of the adhesive and into the PAD. To slow this thermodynamic process even more, the PADs are stored at a cold temperature in the refrigerator.
To determine if the time of storage had any effect on the calculated PAD uptake rates, an experiment was carried out with the same conditions that a standard ASTM MIST would have. A duplicate set of PADs had the adhesive removed to determine if PADs with no adhesive would react similarly during storage. All of the PADs were handled exactly the same way except for the time that they were stored in the refrigerator. The results for this experiment are shown in Figure 7-7. The normal PADs that were extracted immediately had the same uptake rate (3.4 cm/min) as the steady state rate mentioned previously.
6.0
5.0
4.0
3.0
2.0 MeS UptakeMeS Rate (cm/min)
1.0
0.0 Immediate 30 min 1 hr 2 hr 4 hr 1 day 3 day Normal PADs 3.4 3.6 4.1 4.2 3.9 4.7 5.0 No Adhesive 3.1 3.1 3.3 3.2 3.5 3.5 4.1
Figure 7-7. Calculated Uptake Rate (cm/min) Versus the Post-Exposure Storage Time for PADs With and Without Adhesive. All PADs Were Exposed for 30 minutes at 100 mg/m3 and Stored at 4°C.
160 In all cases, the PADs without adhesive adsorbed slightly less MeS and therefore had slightly lower uptake rates. From Figure 7-7 it can be seen that the uptake rates for PADs with adhesive increased significantly during the storage time, from 3.6 cm/min immediately following exposure to
5.0 cm/min three days later. On the other hand, the uptake rates for the PADs that had the adhesive removed stayed fairly constant over most of the storage time. The exception being the three day value which increased to 4.1 cm/min, but it was still lower than the 5.0 cm/min observed for the normal PADs. This slight increase may be attributed to some residual adhesive left on the back of the
PAD that was not completely removed with the isopropanol wipe. From these data, it can be assumed that MeS adsorbed into the adhesive can affect the calculated uptake rate of the PAD, if stored at or above 4°C for more than an hour. It is important to note that the magnitude of the increase in total amount of MeS adsorbed is probably a result of the PADs being completely unprotected and exposed to the full challenge concentration during the test. Being exposed to such a high concentration allows
the adhesive to adsorb more MeS during the test so that during storage more MeS can transfer into the
PAD. Preliminary data from some of the adhesive films that were exposed overnight at 100 mg/m3 showed that the adhesive can adsorb over 190,000 ng of MeS.
Since wrapping the PADs in aluminum foil and storing them at 4°C in the refrigerator did not prohibit the after exposure MeS transfer from the adhesive to the PAD, a second experiment was conducted utilizing different storage temperatures. One easily accessible method for cooling the
PADs to extremely cold temperatures is by packing them in dry ice. At atmospheric pressures dry ice exists as a solid below -70°C, and it can be easily packed onto the PADs in the glass vials while they are being stored or transported. To test the efficacy of dry ice storage, it was compared to the immediate extraction and refrigerator storage. The total amount of MeS adsorbed for each condition is shown in Figure 7-8.
161 80,000
70,000
60,000
50,000
40,000
Amount ofMeS (ng) 30,000 61,009
20,000 43,057 43,138
10,000
0 Immediate 1 Day Dry Ice 1 Day Refrigerator
Figure 7-8. Total Amount of MeS (ng) Adsorbed for PADs Exposed for 30 minutes at 100 mg/m3. PADs Were Extracted Immediately, Stored Below -70°C in Dry Ice for One Day, and Stored at 4°C in a Refrigerator for One Day
As previously determined in the concentration and time exposures, a 30-minute exposure at
100 mg/m3 should produce approximately 43,000 ng on the PAD (Figure 7-5). This amount was consistent with the experiments as seen by the immediately extracted PADs in Figure 7-8. The cold temperature of the dry ice stops any mass transfer from occurring during the one day storage. The total amount of MeS adsorbed (43,138 ng) from the PADs stored in dry ice was almost identical to the immediately extracted amount (43,057 ng). As expected, the PADs that were stored in the refrigerator increased almost 42% with a total adsorption of 61,000 ng of MeS. These data prove that in the event that the PADs cannot be extracted immediately, they should be stored under dry ice or by another means to reach extremely cold temperatures. The main theory behind the colder temperatures
162 is to cool the PADs below the glass transition temperatures of the adhesive and the HDPE membrane
to greatly reduce the risk of any mass transfer from occurring. Further investigation into the most effective temperature needs to be conducted to confirm this theory.
While the dry ice does an excellent job of preventing the desorption process from occurring, it is not the most convenient method to store a large number of PADs, as would be required in an eight subject MIST protocol. If a freezer could reach cold enough temperatures it would be an ideal storage container for a large lot of PADs. To determine if an ordinary commercial freezer would be sufficient, more PADs were exposed and stored under varying temperatures. After an exposure of
120 minutes at 108 mg/m3, the PADs that were immediately extracted contained around 180,000 ng
of MeS. The PADs that were stored in the freezer at an average temperature of -20°C for 1 day had
an average of 190,000 ng, and the PADs stored in the refrigerator at 4°C for one day increased in
MeS amount to 208,000 ng. Therefore, the temperatures attainable in a normal commercial freezer
are not sufficient to prevent the mass transfer from occurring. It is possible that special cryogenic
freezers that can reach even colder temperatures could be used in the place of dry ice. Even though
the temperatures in the freezer did not stop the process completely, it would still be beneficial to store
the PADs in the freezer instead of a refrigerator in the event that they could not be extracted
immediately or packed with dry ice.
7.4 Conclusions
All the data that were gathered show that the best approximation for the steady state film
uptake rate of this lot of Natick Sampler PADs is 3.5 ± 0.5 cm/min. Although this rate falls between
the observed variability of 1.0 – 4.0 cm/min reported for the different areas of the body [58], it is
considerably higher than the 2.0 cm/min indicated by Fedele and Nelson [68]. Also the
163 corresponding average steady state PAD uptake rate is 15.0 ± 1.6 cm3/min, which is considerably higher than the standard rate of 10 ± 2 cm3/min specified in the standard. Since all of the exposed
PADs in this study came from the same lot, it is also highly probable that there is significant variation
between lots. The simple determination of surface area variation from lot-to-lot has shown that there
can be significant differences in the properties of PADs from different lots. Further analysis on the
lot-to-lot variation in the physical properties of the PADs such as film thickness, diffusivity, and
uptake rate may be able to identify any manufacturing problems that exist.
The most significant factors that affect the total amount of MeS adsorbed by the PADs are the
challenge concentration and time of exposure. This study shows that the total amount of MeS
adsorbed follows a highly linear trend with the exposure dosage in the range of conditions that were
tested. The slope of the linear relationship directly corresponds to the PAD uptake rate (cm3/min) and
dividing the slope by the surface area of the PAD yields the film uptake rate (cm/min). The film uptake rate was shown to increase slightly as the challenge concentration decreased. However without further testing at much lower concentrations, no definitive conclusions can be made in regards to how high the uptake rate could increase.
In regards to the variability of the uptake rates, the data show that well defined standard handling methods could eliminate many of the inter-laboratory inconsistencies. PADs used to calculate the lot uptake rate should be exposed for no fewer than 30 minutes, keeping in mind that even longer exposures ensure a steady state condition. In addition, there is no basis for the belief that the PADs saturate in testing conditions that fall below a Ct of approximately 54,000 mg.min/m3.
According to the results, if the PADs cannot be analyzed immediately following an exposure they
should be stored in dry ice or in a cryogenic freezer at extremely low temperatures (-30 to -70ºC).
This practice would help to minimize any transfer of MeS from the adhesive into the PAD.
164 With all things considered, the PADs used in the MIST standard methods from a single lot are very consistent, provided proper techniques are utilized to ensure that the actual amount of the
MeS that diffused through the membrane during the test duration is the amount actually being analyzed. Mass transfer during PAD storage can greatly affect the value that is calculated for the uptake rate. The insights gained from this research can help to improve the MIST standard methods and decrease the variability between laboratories.
165 CHAPTER 8: Investigation of Methyl Salicylate Uptake Rates of Fabrics
Excerpts from the following section were part of a conference report for the 8th International
Meeting on Manikins and Modeling (8I3M) in Victoria, British Colombia in August 2010 and part of a poster presentation at the Chemical and Biological Defense Science and Technology Conference in
Las Vegas, Nevada in November 2011.
8.1 Introduction and Background
A goal of the Center for Research on Textile Protection and Comfort (TPACC) is to make
advances in tests that are used to determine the level of protection a garment provides an individual.
The newly constructed Man-In-Simulant-Test (MIST) facility at North Carolina State University provides both industry and academia the capability to test personal protective equipment with regards to its ability to protect the individual against chemical and biological threats. More importantly this new facility offers a unique opportunity to look deeper into the MIST testing procedures and develop more efficient ways to conduct the test and analyze the results. The outcome of this type of research would be to provide the manufacturers of the protective garments with data that more accurately models the differences in protection between various types of protective ensembles, and ultimately to provide the individuals wearing the garments with the best possible equipment to protect them.
The basic MIST methodology, set forth in ASTM F-2588 [59], requires each test subject to wear thirty Passive Adsorbent Dosimeters (PADs) in anatomical locations specifically chosen to represent the naturally occurring variation in skin across the body. These PADs contain an adsorbent material that collects and traps the chemical warfare simulant vapor. Research has been conducted showing that methyl salicylate (MeS) can be used as an appropriate simulant for sulfur mustard (HD)
166 in regards to determining protection factors [61]. The mass of the MeS detected on each of the PADs
can be factored in with the mass of MeS detected on unprotected or “chamber” PAD to develop a
relative protection factor. Local physiological protective dosage factors are calculated to determine
the protection at each place on the body to provide data on vesicants or blistering agents like sulfur
mustard. On the other hand, nerve agents like VX require a total body accumulation before the
effects are observed so a systemic protection factor for total body exposure is calculated.
One of the main opportunities for advancing MIST technology is the incorporation of
articulated manikins. Testing with human subjects can provide a true-to-life simulation of the
protective equipment, but the variation from person to person requires large subject pools to achieve
statistically significant results. Adapting articulated thermal manikin systems to be used in MIST testing could potentially minimize the issues related to subject variation. The most important challenge involving the inclusion of manikins in the testing is the potential discrepancies between the
MIST results for manikins and human subjects. One theory suggests that with human subjects the simulant can adsorb onto the PAD as well as the subject’s skin or hair. On the other hand, the manikin surface is non-porous so the majority of the simulant that enters the suit will only be adsorbed on the PADs, thus resulting in a lower calculated protection factor. The goal of the current research was to broadly investigate how various textile fabrics adsorb MeS, and to determine if a single fabric could match the uptake rate that is specified for the PADs.
For the first phase of this research, a small bench-scale chamber was constructed and validated (CHAPTER 6: Development of Bench-Scale Methyl Salicylate Exposure Chamber). The smaller dimensions of this chamber allow it to be filled with the MeS vapor and reach equilibrium much more quickly than the full-scale MIST facility; therefore more samples could be exposed to multiple conditions in a shorter amount of time. The current design of the bench-scale MIST
167 chamber is shown in Figure 8-1. The following research shows the feasibility of testing multiple fabrics in the bench-scale chamber to determine if there are differences in their rates of MeS adsorption.
Figure 8-1. Bench-Scale MIST Chamber
8.2 Experimental Methods
8.2.1 Bench-Scale Exposures
8.2.1.1 Bench-Scale Exposure Methodology
The bench-scale parameters used in the testing consisted of 50°C on the MeS boiler, 50°C on
the heating tape, 0.62 L/min of air going through the boiler, 7 L/min of air flowing through the
chamber, 22-23°C chamber temperature, and 52-55% chamber relative humidity. These parameters were used to achieve the MeS concentration of 16 ppm (100 mg/m3) ± 15% stated in the standard
168 [59]. The average concentrations of MeS for each of the three trials were 15.77, 16.47, 16.20 ppm
respectively. The chamber concentration was monitored using a CIC Photonics gas-cell FT-IR with a
MeS calibration range of 1 – 50 ppm. The length of the exposure was fifteen minutes in each trial.
The chamber was allowed to reach equilibrium for one hour prior to introducing samples. After the chamber was opened and a sample was placed inside, the flow from the MeS boiler was increased slightly to bring the concentration back within the acceptable limits. The concentration was monitored during each test and the flows adjusted if necessary.
Five test fabrics were selected for the study, and their properties are given in Table 8-1. The fabrics were cut and sewn into socks that would fit around acrylic cylinders measuring 4” tall and 2” in diameter (Figure 8-2). This sample holder was used so that the fabric would be perpendicular to the flow in the chamber and to simulate how it might fit on a manikin arm or leg. The fabrics were exposed in three trials with two fabrics in the chamber at a time. In each trial, six PADs were included to monitor their adsorption of MeS for comparison across the trials. After exposure, a
2 2 square die (2 /3” x 2 /3”) was used to cut two samples from the fabrics. This process ensured that the
samples going on to further analysis had equal areas of 45.88 cm2.
Table 8-1. Identification and Properties for Fabrics Used in Bench-Scale Exposures
Fabric Weight Fabric Thickness Fabric ID Fabric Description Fiber Type (g/cm2) (mm) Spandex Blend 1 Manikin Sweating Skin Spandex Blend 0.026 1.00 Spandex Blend 2 Synthetic Black Knit Spandex Blend 0.026 0.92 Cotton Jersey Cotton T-Shirt Cotton 0.017 0.74 Polyester Jersey Dacron 64 Jersey Polyester 0.012 0.64 Polyester Interlock Dacron 64 Interlock Polyester 0.022 0.90
169
Figure 8-2. Fabrics and Sample Holders for Bench-Scale Exposures
8.2.1.2 Methyl Salicylate Extraction from PADs and Fabrics
After the PADs were removed from the chamber, they were wrapped in aluminum foil, placed in glass vials, and allowed to sit for thirty minutes in a refrigerator (4°C). Then the Tenax®
TA adsorbent was removed from each PAD through the use of a modified empty 1-mL SPE tube
(Supelco) with a filter in the tip. The SPE tubes containing the adsorbent were then placed into a
Supelco Visiprep DL Vacuum Manifold with autosampler vials to collect the extract. Each SPE tube had two 600-µL aliquots of acetonitrile (ACN) (Fisher Scientific Optima LC/MS Grade, 99.9%) injected and allowed to flow through the Tenax® to extract the MeS. A low vacuum (-2 in Hg) was turned on to drain the last drops out of the tubes, and the vials were capped and stored for later analysis. Previous work on the extraction method showed that approximately 1.054 ± 0.4% mL of solvent would be collected in the vial from the two aliquots introduced. Therefore the solvent volume for all following calculations involving the PAD analysis is 1.054 mL of ACN.
170 Due to the size of the fabric samples, a 3-mL SPE tube was used for the extraction. Each of the fabric squares was rolled up, packed into a tube, and placed on the vacuum manifold. Volumetric flasks (10-mL) were placed in the manifold to collect the extract. Eight successive 1-mL aliquots of
ACN were allowed to flow through each sample and collect in the flasks. A low vacuum was applied to remove any of the remaining extract, and the flasks were then filled to a total volume of 10 mL.
These extract samples were then filtered into vials using 3-mL Luerlok syringes with 0.2-µm pore size Whatman filters attached.
8.2.1.3 Gas Chromatography (GC) Analysis Method
All of the samples from the bench-scale exposures were analyzed on an Agilent 6890N GC with a 7693A Autosampler. Denatured ethyl alcohol (Fisher Scientific) and ACN were used as solvent rinses before and after the 1-µL injections. The inlet was set in the splitless mode and used the Agilent 5138-4711 liner containing glass wool. The inlet temperature was 250°C with a purge flow of 100 mL/min and a purge time of 1.25 minutes. A Restek-1701 column (30 m, 0.32 mm I.D., and 0.25 µm film thickness) was used with a carrier gas flow of 1.0 mL/min of helium, and the analysis was controlled under constant flow. The oven program started at 50°C with a 0.5 minute hold time followed by an increase of 25°C/min to 230°C and ended with a 2-minute hold at 230°C.
The retention time for the MeS peak was 6.722 min. A flame ionization detector (FID) was used at
275°C with hydrogen, air, and make-up gas flows of 30, 400, and 25 mL/min, respectively. The method was calibrated using dilutions of MeS (MP Biomedicals, LLC) in a range of 0.75 – 300 ppm.
171 8.2.2 Full-Scale MIST Exposures
8.2.2.1 Full-Scale Exposure Methodology
Based on the results from the bench-scale exposures, two sets of fabrics with varying spandex
contents were manufactured in North Carolina State University’s weaving laboratory at the College of
Textiles. Polyester was chosen as the base fiber for the first set of fabrics and cotton was chosen for the second set. The identification and physical properties of these fabrics are given in Table 8-2. A circular die with a 2” (5.08 cm) diameter was used to cut samples from each fabric. Using the cutting die ensured that each fabric sample had an equal area (20.26 cm2). Three samples of each fabric were clipped together with paperclips and placed on a hanger in a vertical fashion (each sample hanging just below the previous one) so every sample was completely exposed on all sides. The samples were then placed in the full-scale MIST chamber and exposed to 100 mg/m3 of MeS for 30, 60, and 90
minutes. After exposure the fabrics were placed in individual sealed glass vials to await extraction.
Table 8-2. Identification and Properties for Fabrics Used in Full-Scale MIST Exposures
Weight (g) Fabric Weight Thickness Fabric ID Fiber Ratio Fiber Types Construction (20.26 cm2) (g/cm2) (mm) PS-1 100/0 Polyester/Spandex Jersey 0.26 0.013 0.53 PS-2 95/5 Polyester/Spandex Jersey 0.43 0.021 0.67 PS-3 92/8 Polyester/Spandex Jersey 0.44 0.022 0.69 PS-4 90/10 Polyester/Spandex Jersey 0.43 0.021 0.67
CS-1 100/0 Cotton/Spandex Jersey 0.21 0.010 0.47 CS-2 95/5 Cotton/Spandex Jersey 0.33 0.016 0.59 CS-3 92/8 Cotton/Spandex Jersey 0.35 0.017 0.63
CS-4 90/10 Cotton/Spandex Jersey 0.33 0.016 0.63
172 8.2.2.2 Methyl Salicylate Extraction from Fabrics
A less labor intensive extraction technique was used to remove the MeS from the fabrics
exposed in the full-scale MIST chamber as opposed to the bench-scale chamber. The circular fabric samples were placed in 60-mL glass vials (I-Chem®) that were filled with 25 mL of ACN (Fisher
Scientific Optima LC/MS Grade, 99.9%) and allowed to sit overnight at ambient conditions (no less than twenty hours). Based on previous research focused on the efficiency of MeS extraction from
PADs with ACN, it was assumed that nearly all of the adsorbed MeS would be extracted from the
fabrics during the allotted time. After extraction a 1.5-mL sample of the extract was removed with a
3-mL Luerlok syringe and filtered through a 0.2-µm pore size Whatman filters into an autosampler
vial.
8.2.3 High Performance Liquid Chromatography (HPLC) Analysis Method
The extract samples were analyzed on an Agilent 1260 Series HPLC containing a Binary
Pump SL (Model Number G1312B), a standard autosampler (Model Number G1329B), a thermal
column compartment (Model Number G1613A) and a diode-array detector (Model Number
G4212B). The injector syringe was rinsed in ACN (Fisher Scientific Optima LC/MS Grade, 99.9%)
to prevent carryover, and since the samples were expected to contain high amounts of MeS, the
injection amount was 0.5 µL. An Agilent Poroshell 120 EC-C18 column (3.0 mm x 100 mm, 2.7µm
particle size) heated to 45°C was used with a flow rate of 0.5 mL/min. A 40/60 H2O/ACN isocratic
method was used with a total analysis time of 4.0 minutes. The diode-array detector was set to
monitor at 310 nm.
173 8.3 Results and Discussion
8.3.1 Bench-Scale Exposures
One of the most important variables that must remain constant in order to accurately compare
fabrics is the chamber MeS vapor concentration. There are two ways that the chamber concentration
was monitored to ensure that it was consistent. The graph in Figure 8-3 shows the chamber profiles
from the FT-IR data during each of the 15-minute trials. It is clear that the first 2-3 minutes after the samples were introduced and the chamber was re-closed, the concentration builds back up to within tolerances, and for the remainder of the test it remains at the 16 ppm level. The second method that was used to check the consistency of the MeS concentration between trials was the analysis of the chamber PADs that were included for each trial. The average amount of MeS detected on the PADs was 29,584 ng for the first trial, 29,919 ng for the second, and 29,582 ng for the final trial. The repeatability of the average amount of MeS adsorbed onto the chamber PADs demonstrates the ability to compare the results from following fabric exposures.
Ultimately, the goal of this study is to compare different fabrics’ MeS uptake rates with the
MeS uptake rate for human skin. The PADs were designed to have an uptake rate similar to that found in human skin, specifically the forearm. According to the standard, the MeS uptake rate per area should be between 1.9–2.8 cm/min (10 ± 2 cm3/min with standard area of 4.5 cm2). The uptake rate is calculated according to the equations in ASTM F 2588 [59] (Equation 7-1 in the previous section). The mass of MeS is divided by the average chamber concentration, the exposure time, and the average active sampling surface area of the lot of PADs. The uptake rates for each of the three trials are 3.68, 3.72, and 3.68 cm/min. The consistency among these values shows that the exposure conditions in each trial were very similar, and therefore the fabric results can be compared between the trials.
174 24
22
20
18
16
14
12
10
8
6
Methyl Salicylate Concentration (ppm) 4
2
0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Minutes of Exposure in Chamber
Trial 1 Trial 2 Trial 3 High Limit Low Limit
Figure 8-3. Methyl Salicylate Concentration Profiles for Bench-Scale Exposures
In the first trial, the two synthetic fabrics containing spandex were exposed in the chamber.
The base fibers were not known, but they were assumed to be a type of nylon or polyester. The first spandex blend was from the body suit that was provided by the manikin manufacturer. The second spandex blended fabric was tested to determine if similar fabrics would produce similar results. From the chart in Figure 8-4, it can be seen that both of the spandex blended fabrics had uptake rates around seven times larger than that of the PADs. Also, this trial showed that similar fabrics may show similar trends when it comes to their uptake of MeS. The second trial consisted of a cotton fabric and a polyester fabric both having a jersey construction. The fabric properties given in Table 8-2 show that these fabrics were very similar in weight, thickness, and construction so any observed differences
175 should be due to the fiber type. The uptake rates for the cotton and polyester fabrics were 0.31 and
0.20 cm/min respectively. These rates are close to an entire order of magnitude lower than the chamber PADs. The third trial only contained the polyester interlock fabric, and it also yielded a low uptake rate of only 0.15 cm/min. From the second and third trials, it appears that MeS does not readily adsorb onto the cotton and polyester fibers. The only other option is that the fibers hold the simulant tighter, and it may be necessary to employ a move strenuous extraction method. However, the first trial did prove that the extraction method used could remove a large amount of the simulant.
Since the fabrics without spandex did not adsorb a large amount of MeS, it is safe to assume that the spandex is responsible for the extremely high adsorption in the first trial.
30 27.62
24.69 25
20
15
MeS Uptake Rate (cm/min) Rate Uptake MeS 10
3.68 3.72 3.68 5
0.31 0.20 0.15 0 Trial 1 PADs Trial 2 PADs Trial 3 PADs Spandex Blend 1 Spandex Blend 2 Cotton Jersey PET Jersey PET Interlock
Figure 8-4. Methyl Salicylate Uptake Rates for PADs and Fabrics
176 8.3.2 Full-Scale Exposures
From the knowledge gained in the bench-scale exposures, two sets of fabrics with polyester and cotton base fibers were constructed with a range of spandex (0–10%) to determine the effect of the spandex content on the total adsorption of MeS. These fabrics were exposed in the full-scale
MIST chamber so that all of the samples could be tested at the same time instead of in separate trials.
The results for the total amount of MeS adsorbed on each fabric are shown in Figure 8-5 with the appropriate spandex contents labeled. The most important observation that can be made from the figure is that the inclusion of spandex greatly increases the total amount of adsorbed MeS. The 100% polyester and 100% cotton fabrics, in agreement with the bench-scale exposures, adsorbed relatively little MeS. However, the inclusion of only 5% spandex increased the adsorption nearly an order of magnitude for both fabric types. Increasing the spandex content to 8% and 10% increased the total adsorption approximately double that of the 5% spandex fabric. Very little difference was observed between the 8% and 10% spandex fabrics which may be attributed to small difference in spandex content between them. It is logical to conclude that a fabric with 15% or 20% spandex could adsorb three to four times the amount of MeS as the fabrics with 5% spandex.
A second observation from this figure is that the fabrics seem to nearly saturate with MeS after only thirty minutes of exposure. Some increase in the total adsorbed amount of MeS can be seen during the sixty and ninety-minute exposures, but the rate is extremely lower than what would be expected for a PAD or for human skin. The calculated uptake rates (cm/min) for each of the fabrics are given in Figure 8-6. Since the fabrics became saturated after the thirty-minute exposure and because a standard ASTM test is only thirty minutes, the data from the sixty and ninety-minute exposures have been excluded from the figure. The variability in the uptake rate observed in different body regions (1.0 - 4.0 cm/min) and the value for the forearm and PAD (2.0 cm/min) are given.
177
PS-1 PS-2 PS-3 PS-4 CS-1 CS-2 CS-3 CS-4 700,000 8% and 10% Spandex 600,000
500,000
400,000
5% Spandex 300,000
200,000
100,000 0% Spandex Total Amount of Methyl Salicylate Adsorbed (ng) Adsorbed Salicylate of Amount Methyl Total
0 20 30 40 50 60 70 80 90 100 Minutes of Exposure at 100 mg/m3 .
Figure 8-5. Methyl Salicylate Adsorption of Spandex-Blended Fabrics
178 10.0
9.0 8.9 8.5 8.6 8.2 8.0
7.0
6.0 Uptake Rate Range of Specified Uptake Rate of Human Skin Forearm and PAD 5.0 4.2 4.0
MeSUptake Rate (cm/min) 3.5 3.0
2.0
1.0 0.2 0.4 0.0 100% Polyester 95/5 92/8 90/10 100% Cotton Jersey 95/5 92/8 90/10 Cotton Jersey Polyester/Spandex Polyester/Spandex Polyester/Spandex Cotton/Spandex Cotton/Spandex Spandex Jersey Jersey Jersey Jersey Jersey Jersey
Figure 8-6. Measured Uptake Rates of Various Fabrics After 30 minutes of Exposure to 100 mg/m3
179 As can be seen from Figure 8-6, only the 5% spandex fabrics have uptake rates similar to those observed in the various body regions. None of the tested fabrics is significantly close to the 2.0 cm/min value that has been specified for the PADs, but the value for the 95/5 polyester/spandex fabric agrees very well with the PADs that were used in the bench-scale exposures (3.68 cm/min). It is reasonable to believe that a fabric with 2-3% spandex may achieve a fabric uptake rate closer to the suggested value.
8.4 Conclusions
The main goal of this research was to offer an exploratory view into the MeS uptake rates of fabrics. From the bench-scale exposures it was concluded that the fabrics could be exposed and compared across trials, and that spandex contributed significantly to the total adsorption of MeS.
Also it can be concluded that the existing manikin body suit adsorbs a significant amount of MeS and has the potential to interfere with the MIST results if it covers the manikin during a test.
The various spandex-blended fabrics exposed in the full-scale MIST chamber reinforced the hypothesis that the MeS adsorption increases with the increasing spandex content. Fabrics containing
5% spandex had the closest uptake rates to those observed across the human body. The 8% and 10% spandex fabrics had much higher uptake rates and capacities for MeS than other fabrics that were tested. Therefore, it can be concluded that fabrics that contain greater than 10% spandex have a significant chance to affect the results of MIST testing if they are worn under the ensemble and should be avoided. These exposures also revealed that 100% cotton and 100% polyester jersey fabrics adsorb very little MeS, and therefore can be used as undergarments in the MIST protocols without the possibility of interfering with the test results.
180 CHAPTER 9: Investigation into the Variability of Man-In-Simulant-Test Results with Human
Subjects and Feasibility of Incorporating Articulated Manikins
Excerpts from the following section were part of a conference report for the 5th European
Conference on Protective Clothing (ECPC) and the Nordic Coordination Group on Protective
Clothing as a Technical Preventive Measure (NOKOBETEF) 10 in Valencia, Spain in May 2012.
9.1 Introduction
Since it was developed by the U.S. Army in the early 1990s [58], the Man-In-Simulant-Test
(MIST) has been the primary method used to evaluate full ensembles with regards to their chemical protective performance. MIST evaluations involve exposing human test subjects wearing full protective ensembles to a low toxicity simulant for chemical warfare agents. The target property that is assessed by the MIST is the ensemble’s ability to prohibit or minimize inward leakage of the simulant vapor through interfaces and closures such as seams, seals, or zippers. The amount of simulant that enters the ensemble is measured using Passive Adsorbent Dosimeters (PADs) placed in specific locations across the body. These PADs are collected after the exposure, and the mass of the simulant on each PAD is processed through a Body Region Hazard Analysis (BRHA) to generate both localized and systemic protection factors for the ensemble.
The required exposure conditions, subject protocols, and calculation methods for the MIST are specified by two distinctly different standards. The American Society for Testing and Materials
(ASTM) regulates the ASTM F 2588 MIST standard [59] which is intended to be used to evaluate
protective ensembles that first responders may use. This method exposes at least four human subjects
in the MIST chamber for thirty minutes at a simulant concentration of 100 mg/m3. The second
181 standard is maintained by the U.S. military and is referred to as Test Operations Procedure (TOP) 10-
2-022 [60]. Since the TOP method is intended to evaluate military ensembles that may be used for
extended periods of time, it requires that the human subjects be exposed to the same concentration of
the simulant (100 mg/m3) for two full hours.
Regardless of the standard method that is followed, the use of human test subjects is a critical component to achieving an accurate “true-to-life” assessment of the ensemble’s protective performance. However anytime human subjects are used in a test, there will inherently be a degree of variability that is introduced. The variation in body size and shape can affect the fit of the garment between different subjects, and the day-to-day differences in body temperature, sweat rate, and overall physiology may affect the results from replicates of the same subject. In an attempt to overcome this variability the ASTM standard requires at least four test subjects and two different sizes of the ensemble to be evaluated [59], while the standard TOP procedure requires eight subjects to be tested [60]. Using four to eight subjects to assess each ensemble generates a large number of samples that can require multiple days to analyze.
In addition to the variability issues, another challenge with using human subjects is the logistics involved in conducting the tests. One issue is that all human subject testing must be reviewed by an institutional review board and deemed to be ethical prior to conducting the tests.
Depending on the institution, this process could take weeks or months before testing could be started.
Another issue involved with human subject testing is the selection and scheduling of the appropriate subjects. Lastly, human subjects can only be subjected to a range of conditions deemed safe by the review board, but some ensembles may need to be evaluated at extreme temperatures or durations.
One method that could potentially alleviate the need to test so many human subjects is to incorporate articulated thermal manikins into the MIST methodology. Thermal manikins, like the one
182 shown in Figure 9-1, provide the same subject body size for each test and are capable of maintaining a consistent surface temperature, sweating rate, and walking rate. Incorporating manikins into chemical protective performance testing may also open up the possibility of evaluating the ensemble’s performance against live agents instead of only simulants. Exposing human subjects to live agents is deemed unethical and far too hazardous; therefore a manikin that has been shown to generate results comparable to human testing may provide a means to assess the ensemble in an even more realistic manner.
Figure 9-1. NEWTON Sweating Thermal Manikin at North Carolina State University
183 There has been very little research conducted and published comparing the MIST results between individuals or between humans and manikins. Some of the only work in this area has been conducted jointly by the Royal Military College (RMC) in Canada and the Netherlands Organization for Applied Scientific Research (TNO), where a basic comparison of human subjects and manikins was performed [77]. The major outcome of this investigatory project was that a significant difference between the two data sets was observed. The geometric averages of the local protection factors for the two ensembles that were tested on both human subjects and manikins are shown in Figure 9-2
(higher PF values correspond to a higher level of protection). Since the human subjects were tested at
RMC in Canada and the manikin evaluations were conducted at TNO in the Netherlands, a second set of manikin tests were conducted at RMC to eliminate the possibility that the differences in results were due to testing chambers or conditions. The second manikin tests, conducted with a generic department store non-moving manikin, showed that the results between humans and manikins could be similar and within the normal deviations that occur. The researchers determined that the most likely source of the inconsistencies were due to differences in the activity level and the non- anthropomorphic shape of the manikin.
A follow-on research project, conducted by RMC, focused on developing a manikin that could provide the anthropomorphic body shape and realistic movements as well as a compressible skin-type material that would allow for better seals around the waist, wrists, and head [78]. This next generation manikin system was developed in conjunction with a company in the United Kingdom called i-bodi Technology LTD. A comparison of four human subjects and four manikin tests showed that the protection factor results were much more consistent with the new manikin design [78].
184
Figure 9-2. MIST Results from RMC and TNO Human-Manikin Comparison [77]
The Center for Research on Textile Protection and Comfort (TPACC) at North Carolina State
University (NCSU) constructed a MIST facility in 2008. Being located at an academic institution has allowed for more in-depth research to be conducted on the MIST method itself. Since TPACC has a history of using manikins in flash-fire exposures and thermal/comfort testing, a natural area of interest is the incorporation of manikin systems into the chemical testing field. Whereas the previous research projects focused on developing a new manikin system and comparing the average protection values between humans and manikins, TPACC has conducted an explorative study focused around
185 assessing the variability of MIST results from the human subject protocol and determining the feasibility of testing with manikins.
9.2 Experimental Methods
9.2.1 Methods Used to Adapt NEWTON Manikin for MIST Testing
Due to the high cost of the manikin that was developed by i-bodi Technology LTD. and the availability of the existing manikins in TPACC, a NEWTON type manikin developed by
Measurement Technology Northwest was chosen to be used for the comparison. The standard
NEWTON manikin is constructed of a carbon-fiber/epoxy composite and is sized to reflect the body dimensions of the fiftieth percentile Western or Asian male body [79]. The NEWTON manikin has thirty-two active heating zones that are capable of maintaining the body surface at a temperature similar to the average human skin temperature. The manikin is also equipped with over one hundred sweat pores that allow for a controllable sweating rate, and a fabric body suit or sweating skin provides even distribution of the sweat across the manikin surface. NEWTON manikins have articulated joints in the shoulders, elbows, hips, knees, and ankles that allow for garment donning and a basic walking motion. The particular NEWTON manikin used for this study was equipped with a mechanism to simulate the breathing process through either the nose or mouth [79].
One of the main issues with incorporating a manikin into the MIST protocol is that the manikin has a support post located on the top of it head which is used to connect it to the walking stand. To allow the chemical protective ensembles to fit on the manikin, a hole had to be cut into the suit that would allow the hood to slide down over the head post. Introducing an opening in a chemical protective ensemble is not desired, so after the hood of the garment was put into place, a
186 PTFE sealant was wrapped around the opening to seal the fabric to the head post. The seal on the manikin head post is shown in Figure 9-3.
Figure 9-3. PTFE Sealant on the Manikin Head Post
The movement and range of motion provided by the commercially available NEWTON manikin is a very basic and restricted to a walk or shuffle. For the purposes of MIST evaluations, it is highly important to stress the interfaces of the garment so that the integrity of the seal or closure mechanism can be tested. These stresses occur in the human subject protocol through the use of the
187 various exercises. In order to give the manikin a more realistic or natural range of motion, it was connected to an elliptical exercise walker that was driven by the motor from the manikin’s walking stand (Figure 9-4). The motion provided by the elliptical walker allowed for the shoulders, elbows, hips, and knees to all flex and put stress on the garment as opposed to standard walking motion that only flexes the shoulders and hips. It was not desirable to make holes in the garment to attach the feet and hands to the elliptical walker, so the physical attachments shown in Figure 9-5 were fabricated.
These attachments were designed to allow for a quick and easy connection when the manikin was brought into the MIST chamber. Also the manikin’s joints were specifically designed for ease of replacement due to the extra wear that can occur from the extended range of motion.
Figure 9-4. MIST Manikin on Elliptical Walker in NCSU’s MIST Facility
188
Figure 9-5. Physical Attachment Points Between Manikin and Elliptical
189 9.2.2 MIST Exposure Methodology
To assess the variability between human subjects and between humans and manikins, MIST
trials were conducted in the NCSU MIST facility. To ensure that the exposure duration, test conditions, and movements were as similar possible, all trials followed a modified version of the
ASTM F 2588 standard. Since the manikin was adapted to use the elliptical walker, the human subject protocol was modified so that the subjects also used the same type of elliptical equipment.
The exercise protocol and test conditions for both manikin and human subject testing are given in
Table 9-1.
Table 9-1. Modified MIST Exercise Protocol for Human Subjects and Manikin
Test Condition or Parameter ASTM F 2588 Exercise Duration
27 ± 5°C Temperature Standing Rest 5 minutes (80 ± 10°F)
% Relative Humidity 65 ± 20% Elliptical Walker at 1 mph 25 minutes
Methyl Salicylate Conc. 100 ± 15 mg/m3 Standing Rest 5 minutes 1.55 ± 0.65 m/s Wind Speed Elliptical Walker at 1 mph 25 minutes (3.5 ± 1.5 mph)
Number of PADs 30 Standing Rest 5 minutes
Test Duration 65 minutes
The initial and final five-minute rests were included due to the time that it took to get the
manikin in the chamber and attached to the elliptical walker. The test duration was increased to sixty- five minutes to allow for the manikin to complete two cycles of walking and resting. The attached
190 motor was capable of driving the manikin’s movement at approximately 1 mph. Therefore the
subjects were instructed to keep their pace close to the manikin’s by watching the screen on the
elliptical walkers. Since the manikin was not able to be put into a sitting position, the subjects were
asked to remain standing during the rest period. The human subjects’ entrance into the chamber was
staggered by thirty minutes so that two subjects were in the chamber at all times. A photo of one of
the actual human subject tests is shown in Figure 9-6. Prior to and during the test, the manikin surface temperature was maintained at 35°C to reflect an average skin temperature of a human being as stated in ASTM F 1291 Standard Test Method for Measuring the Thermal Insulation of Clothing
Using a Heated Manikin [80].
Figure 9-6. Human Subjects in MIST Trial for Blauer® MIRT Class 3 Ensemble
191 To assess the variation between human subjects, three eight-subject MIST trials were conducted using the Blauer® WZ9430 MIRT ensemble with an FR-M40 full-face respirator as shown in Table 9-2. This ensemble was selected because it was certified to NFPA Class 3 specifications which provide a moderate to low level of vapor protection. A lower-level of vapor protection ensured that the simulant would infiltrate the ensemble and comparisons between subjects could be made.
Under the ensembles, the subjects wore 100% cotton t-shirts, boxer-briefs, and socks. The trials consisted of testing the same eight subjects in the same ensemble on three separate occasions. After each trial, the suits, masks, and all other equipment were allowed to air dry on a garment dryer overnight. The three manikin MIST trials were conducted with three brand new Blauer® MIRT ensembles so direct comparisons could be made.
For the human subject testing, a fourth MIST trial was conducted with six of the same human subjects wearing the LION ® ICG ensemble with the FR-M40 full face respirator as shown on the left side of Table 9-2. This ensemble was selected because it was certified to the NFPA Class 2 specifications that provide a high level of vapor protection. The purpose of this extra trial was to provide data on an ensemble that was supposed to have higher protection values.
All human test subjects were selected from the City of Raleigh Fire Department and were
HazMat certified. According to NCSU protocol, all human testing was reviewed and approved by an
Institutional Review Board for ethical purposes.
All systemic and localized protection factors were calculated according to the method provided in ASTM F 2588 and also given in Appendix 10.3.2A.1. All results referred to as average values were calculated based on the geometric means for the lognormal distribution.
192 Table 9-2. Ensembles Used in Human-Manikin MIST Comparisons [54] [55] Blauer ® LION® ICG WZ9430 MIRT
9.2.3 PAD Extraction Methodology
An extraction method developed at NCSU was employed to recover the methyl salicylate
(MeS) from the PADs. After exposure, the adsorbent was removed from each PAD through the use of a vacuum and a modified empty 1-mL SPE tube (Supelco) with a filter in the tip. The SPE tubes containing the adsorbent were then placed into a Supelco Visiprep DL Vacuum Manifold with autosampler vials to collect the extract. Each SPE tube had two 600-µL aliquots of acetonitrile
193 (ACN) (Fisher Scientific Optima LC/MS Grade, 99.9%) injected with a repeater pipet that had a 10- mL capacity pipet tip. The ACN was allowed to flow through the adsorbent to extract the MeS.
A low vacuum (-2 in Hg) was turned on to drain the last drops out of the tubes, and the vials were capped and stored for later analysis.
9.2.4 High Performance Liquid Chromatography (HPLC) Analysis Method
The PADs were analyzed on an Agilent 1260 Series HPLC containing a Binary Pump SL
(Model Number G1312B), a standard autosampler (Model Number G1329B), a thermal column
compartment (Model Number G1613A) and a diode-array detector (Model Number G4212B). The
injector syringe was rinsed in ACN (Fisher Scientific Optima LC/MS Grade, 99.9%) to prevent
carryover, and the injection amount was 4 µL. For highly concentrated specimens, the injection
amount was decreased to 0.5 µL in order to avoid overloading the detector. An Agilent Poroshell 120
EC-C18 column (3.0 mm x 100 mm, 2.7µm particle size) heated to 45°C was used with a flow rate of
0.5 mL/min. A 40/60 H2O/ACN isocratic method was used with a total analysis time of 4.0 minutes.
The diode-array detector was set to monitor at 310 nm.
9.3 Results and Discussion
9.3.1 Variability in MIST Results from Multiple Human Subjects
One of the primary goals of this research was to determine the magnitude of variability that
can be expected in standard MIST trials using human subjects. Therefore, three MIST trials were
conducted using eight subjects for a total of twenty-four ensemble evaluations. The Blauer® MIRT
ensemble was chosen to use in the comparison because it was a certified NFPA Class 3 Ensemble,
194 and would provide a moderate level of vapor protection. Each of the eight subjects was tested three
times in their ensemble, and the logarithmic plot of the overall average systemic and localized PPDF values are given in Figure 9-7, the dotted line indicates the passing local PPDF value (120) for a
NFPA Class 3 Ensemble [50]. For reference purposes, the larger PPDF values correlate to better overall protection. Since the protection factors are ratios, they generally follow a lognormal distribution and therefore, the geometric mean and geometric or multiplicative standard deviation should be used to compare results as opposed to the arithmetic mean and standard deviation. The results in Figure 9-7 show that the calculated PPDF values across the various body regions follow a very similar trend for each of the eight subjects.
Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Subject 7 Subject 8 100000
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Figure 9-7. Geometric Mean PPDF Values for 8-Subject MIST Trials on the Blauer® MIRT Ensemble (Average of Three Replicates for Each Subject)(Dotted Line Indicates the NFPA 1994 Passing Local Protection Factor (120) for a Class 3 Ensemble)
195 There are a few areas such as the forearms or abdomen that have slightly higher degrees of variation, while other areas around the crotch and lower legs agree very well among subjects. It is noteworthy that nearly all of the body regions had protection factors that would be considered a pass by the
NFPA 1994 criteria.
While the magnitude or absolute value of the protection is very important for certification purposes, the variability in that value is of more interest for this research, in particular, the variability among the three replicate MIST trials and the variability associated with the number of subjects that are tested. The degree of variability for this study has been gauged by the range of the 95% confidence intervals. If the confidence interval is extremely large, it is difficult to conclude with high degree of certainty that the true protection value is near the measured quantity. The geometric mean of the systemic PPDF values for each trial is given in Figure 9-8 with the 95% confidence intervals indicated by the error bars. The data show a slight decrease in the overall protection from the first trial to the third, which was also observed in the PPDF values for many of the local body regions.
This decrease is most likely due to the fact that the exact same ensembles were tested in each trial.
By the third trial, the fabric of the ensemble may have adsorbed some of the simulant vapor and released it while the ensembles were being testing. This finding shows that it is best to test new garments that have not been previously exposed. As was the case in this research, the cost and manufacturing time of the ensembles may not always allow for new garments to be used. In the event that previously exposed garments have to be used for MIST trials, it is imperative to check the level of simulant that may be released by the fabric prior to testing. This check can be conducted by placing the garment in a sealed container or bag along with three or four PADs to measure the simulant concentration given off by the ensemble’s fabric.
196 With regards to the variability among the trials, the first replicate had an average systemic
PPDF value of 276 + 48% and - 33%, the second trial had a value of 248 + 19% and - 16 %, while the third trial had a value of 139 + 39% and - 28%. The first and third trials had similar ranges of confidence, while the second trial had a slightly smaller confidence interval. The smaller range for the second trial was a result of not having an extremely high or extremely low value as opposed to the other two trials. The similar and relatively small degree of variation among the three trials suggests that eight subjects are sufficient to evaluate an ensemble with a high degree of confidence.
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1 Blauer MIRT Rep 1 Blauer MIRT Rep 2 Blauer MIRT Rep 3
Figure 9-8. Comparison of Geometric Mean Systemic PPDF Values for Each MIST Trial with the Blauer® MIRT Ensemble (Error Bars Indicate the 95% Confidence Interval)(Dotted Line Indicates the NFPA 1994 Passing Systemic PPDF (76) for a Class 3 Ensemble)
197 To test this hypothesis, the geometric means and confidence intervals for the first MIST trial
(Rep #1) were calculated using the results from two, three, four, five, six, seven, and all eight subjects. The results of these calculations are shown in Figure 9-9. As expected, the confidence intervals, and therefore the variability, decrease as more subjects are used to evaluate the ensembles.
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1 2 Subjects 3 Subjects 4 Subjects 5 Subjects 6 Subjects 7 Subjects 8 Subjects
Figure 9-9. Effect of Number of Subjects on the Systemic PPDF Values for the Blauer® MIRT Ensemble (Error Bars Indicate the 95% Confidence Interval)
The lowest amount of variation is observed when eight subjects are used, which reinforces the requirement on the number of subjects for the TOP method. The data calculated using only four subjects show that the requirement of at least four subjects in the ASTM method most likely provides an accurate measure of the true protection value. The extremely large confidence interval on the data
198 calculated with two subjects proves that even though the average protection value was very close to
that calculated from all eight subjects, it is highly likely that if only two subjects are used, one or both
of them may generate a protection factor unreasonably high or low. From the data, the best
recommendation that can be made on the number of subjects required to achieve a relatively high
degree of confidence in the systemic PPDF values is at least four to eight subjects.
A second ensemble was evaluated to determine if the level of protection affected the
variability that was observed. Since the previous trials used an NFPA 1994 Class 3 ensemble that
provided moderate vapor protection, the LION® ICG garment was selected as a comparison
ensemble because it was certified to NFPA 1994 Class 2 criteria to provide a much higher level of
vapor protection. Due to scheduling conflicts, only six of the original human subjects were available
for the second set of MIST trials. The logarithmic plot of the average systemic and localized PPDF
values for the six subjects in both ensembles is given in Figure 9-10 with the 95% confidence intervals. As expected, the Class 2 ensemble provided a significantly higher level of protection, with an average systemic PPDF of 817 + 46% and - 32%. This confidence interval is very similar to that observed in the systemic values for the six subjects tested in the Blauer® garment (+ 57% and - 36%).
While the confidence intervals for the systemic values are very similar, it can be seen from
Figure 9-10 that there is considerable difference in the localized PPDF values for specific body regions. In particular, most of the values around the neck, the torso, and the arms are much more variable in the Class 3 ensemble as opposed to the Class 2 ensemble. One possible explanation for this difference is that an ensemble that provides a higher level of protection may lead to less variation in the MIST results. Since the Class 2 ensemble is intended to provide a higher level of vapor protection, it was designed as a one piece garment with the glove and boot liners built into the garment material. The only area that is available for leakage to occur is in the area around the neck
199 which has an air-tight zipper to attach the garment hood. On the other hand, the Class 3 ensemble is
not intended to provide an exceptionally high level of vapor protection. Therefore it was designed as
a two-piece ensemble with one zipper connecting the jacket and trousers and another zipper that is used to seal the jacket and hood around the respirator. The ensemble also uses gloves and glove liners that are fully detachable from the sleeves of the jacket. These design features make the Class 3 ensemble more difficult to dress and appropriately seal each time it is worn. If the hood and jacket are not fully sealed around the respirator face piece, then a potential area for leakage can occur. The same principle applies for the straps around the wrist that are used to seal around the glove liners.
Blauer MIRT Ensemble LION ICG Ensemble 100000
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Figure 9-10. Comparison of Geometric Means of PPDF Values for 6-Subject MIST Trials on Blauer® MIRT Class 3 Ensemble and LION® ICG Class 2 Ensemble
200 Since the Class 2 ensemble is a one-piece suit, it is much easier to ensure that it is worn and sealed appropriately each time. In light of this theory, it is no coincidence that the body regions that experienced higher levels of variation in PPDF values were located in close proximity to the areas with the differing design features. Further research may be able confirm that the observed variability in protection values may point to areas of weakness in the garment design.
9.3.2 Feasibility of Using a Manikin in MIST Testing
Since the previous research projects conducted by RMC and TNO had already shown that
MIST testing with manikins could produce protection factors similar to those generated with human subjects, the purpose of the NCSU investigation was to determine if manikin testing could feasibly be used in the place of human subject testing. The appeal of using manikins instead of human subjects is mainly focused on prototype ensemble evaluations and is centered around the idea that manikin testing could be conducted to achieve the same level of evaluation with fewer replicates which would result in fewer samples, less analysis time, and a lower cost of testing. Therefore using manikins instead of humans subjects would only be deemed feasible if these assumptions were proven to be accurate.
Since standard methods that currently use manikins, such as ASTM F 1291, require all manikin measurements to be conducted three times [80], an important question is whether or not three replicates of manikin testing can provide similar variability and confidence intervals to that of an eight-human subject MIST trial. The results from the first eight-human subject MIST trial on the
Blauer® Class 3 Ensemble are compared to the geometric mean values from three manikin evaluations in Figure 9-11 with the 95% confidence intervals indicated by the error bars. The first observation that can be made is that the overall trend of protection across the different body regions can be considered fairly similar between the human and manikin tests, reinforcing the findings by
201 RMC and TNO that manikins do generate protection factors in the same range as human subjects [77]
[78]. The second and most striking observation that can be made from the two sets of data is that the
PPDF values calculated from the manikin trials have a significantly higher degree of variability and therefore, result in a lower degree of confidence in the measured protection when compared to the eight human subjects. Consequently, it can be concluded that three replicates with manikins do not produce results with similar variability or degrees of confidence as eight human subject MIST trials.
Blauer MIRT 8-Human Subjects Blauer MIRT 3-Manikin Subjects 100000
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Figure 9-11. Comparison of Geometric Mean PPDF Values for an 8-Human Subject and a 3- Manikin Subject MIST Trial on the Blauer® MIRT Class 3 Ensemble (Error Bars Indicate the 95% Confidence Intervals)
202 It has been shown that eight human subjects can provide an evaluation with a smaller degree of variability than three manikin tests, and the data in Figure 9-12 show that a MIST trial using six, four, or only three human subjects can still provide less variable results than using three manikin replicates.
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1 3 Manikin 3 Human 4 Human 6 Human 8 Human
Figure 9-12. Effect of Number of Human Test Subjects on the Variability in Systemic PPDF Values Compared to the Geometric Mean of Three Manikin Tests on the Blauer® MIRT Ensemble (Error Bars Indicate the 95% Confidence Interval)
While the manikin results do appear to be more variable, a comparison of the three manikin tests with the three replicates of each of the eight human subjects offers a slightly different take on the findings. This comparison, given in Figure 9-13, shows that Subjects 1 and 8 exhibited similar
203 variability to that of the manikin and suggests that it may be possible for eight replicates with the
manikin to provide results more similar to the eight human subjects. By conducting three MIST trials
with eight manikin replicates, future research could clarify this theory.
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1 Manikin Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Subject 7 Subject 8
Figure 9-13. Geometric Mean Systemic PPDF Values for Three Replicates of Manikin and Human Subject MIST Trials on Blauer® MIRT Class 3 Ensemble (Error Bars Indicate 95% Confidence Interval)
9.4 Conclusions
In summary, standard MIST evaluations involving human subjects do have a significant degree of variability associated with the results. Whether this variability is due to the differences among the subjects’ physiology, the ability to appropriately seal the interfaces in the garment, or
204 simply part of the test methodology are viable areas for future research. The MIST trials on the
Blauer® ensemble showed that the standard requirements to evaluate four to eight human subjects are
justified and should be consider critical variables to evaluate an ensemble to the most accurate degree
possible. It should be noted that during research and development of new chemical protective
ensembles fewer subjects could be used gauge the protection level of the prototypes, keeping in mind
that more subjects must be used for certification purposes.
In regards to development of ensembles, this research has shown that an important aspect of designing the interface areas such as closures around the respirator, neck, hands, or wrists, is to consider whether or not the closure mechanism can be sealed appropriately each time the garment is donned. Along the same lines, it may be possible to determine how well a closure technology works not only by the magnitude of the protection factors but also by the degree of variability that is observed.
The results from the manikin MIST testing have shown that the appeal of using manikins
instead of human subjects to save time and money is a theory that is not grounded in facts. Whether
more manikin replicates can produce less variable results is of little practical value to this research
because the results have shown that conducting three manikin tests instead of four or eight human
subject tests is not a feasible alternative. To achieve a similar degree of confidence in the measured
protection values, the manikin, most likely, will have to be evaluated the same number of times as
human subjects.
In addition, the logistical issues associated with manikin testing further discourage the
replacement of human subjects. The manikin is much more difficult to dress in the chemical
protective ensembles than the human subjects, and it can provide little to no feedback as to whether
the ensemble has been appropriately donned and sealed. The normal procedure to conduct a MIST
205 trial with human subjects consists of evaluating up to four subjects in the chamber at one time and can
be extended to allow for testing up to eight subjects in one day. Unless a facility invests a significant
amount of money into purchasing multiple manikins or conducts a considerable amount of research to
determine if a manikin can be sufficiently decontaminated after a test, only one to two manikin tests
can be conducted in one day.
Considering all of the advancements that have recently been made in the MIST methodology
by the research conducted at NCSU, a standard MIST trial with eight human subjects can be
conducted, analyzed, and reported in as little as one week. This rapid turnaround with human
subjects makes the idea of incorporating a manikin that is more variable, more time consuming, and less accurate than human subjects highly undesirable.
The best recommendation that can be made for future manikin research is in the area of live agent testing. Human subjects will never be used in this capacity, and the results from the manikin testing at RMC, TNO, and NCSU have all shown that using a correctly designed manikin with an appropriate number of replicates can evaluate the protective performance of an ensemble to a reasonably accurate degree.
206 CHAPTER 10: Research Conclusions, Recommendations, and Proposed Future Research
10.1 Summary of Research and Overall Conclusions
The studies that were conducted as part of this research project were focused on not only
understanding and questioning the current MIST methodologies but providing the ASTM and military
standard committees with tangible, published results to improve and advance the MIST as a whole.
The overarching conclusion that can be made by combining all of the results from the research is that
the human protocol for MIST evaluations, while having an inherent degree of variability, does exactly
what it was designed to do as long as caution is taken to ensure that all of the procedures are
conducted appropriately and consistently. The MIST method is still the best approach that can be
used to evaluate the protective performance of full CBRN ensembles.
10.2 Research Conclusions
10.2.1 Conclusions Pertaining to the Advancement of the MIST Methodology
One of the main objectives of this research was to develop a more complete understanding of
where the MIST originated, why it is used to evaluate ensembles, what information the MIST results
can offer about an ensemble’s protective performance, and how the calculations relate to the overall protection. The review of literature that was conducted and is included in Chapters 2-4 provides an extensive background that answers many of these questions. These chapters along with the research results, can serve and an informative manual to better explain many aspects of the MIST for academia, industry, government, or military personnel that are involved in CBRN ensemble development or MIST evaluations. If the personnel working in this field are better educated and
207 informed on MIST method itself, they will be able to fully understand the results of the trials, gather
more in-depth information about the tested ensemble, and ultimately make better protective ensembles in a shorter amount of time.
Since neither of the MIST standards addresses the extraction and analysis of the PADs, the development of appropriate techniques that quickly and efficiently determine the amount of adsorbed
MeS was a major goal for this research. The most important conclusions that can be made for this phase of the research are that the NCSU extraction and analysis methods provide over 98% extraction of MeS from the adsorbent, can be detected and quantified over the full range of expected MeS amounts, and allow for the turnaround of a standard eight subject MIST trial in as little as one week.
A third highly important objective of the research was to fully characterize the PADs according to both their physical properties and the effect of the exposures conditions. It can be concluded that PADs from the same lot are relatively similar in their active sampling surface areas and in their measured uptake rates. A significant degree of variability does exist between the PADs from different lots which can potentially be attributed to differences in the thickness or diffusivity of the HDPE film. The PADs also showed the expected linear relationship between the total adsorbed
MeS and the exposure dosage. Since it can be concluded that the PADs will not saturate if fully exposed during a standard thirty-minute or two-hour test, it can be recommended that all chamber
PADs be exposed for at least thirty minutes to achieve a steady state uptake rate.
The investigation into the PAD storage and handling procedures showed that both the storage duration after exposure and the storage temperature can drastically affect the measured amount of
MeS on the PADs. This observation can most likely be attributed to mass transfer of MeS from the adhesive and into the PAD during storage. The best recommendation that can be made from these
208 findings is to ensure that the PADs are extracted within one to two hours after being exposed or to
store them in extremely cold temperatures, such as in dry ice, to prohibit any mass transfer.
Overall, the characterization study showed that within the same lot, the PADs are not nearly
as variable as once thought, if appropriate measures are taken to ensure that the PADs from one
subject are exposed, stored, extracted, and analyzed in the same manner as all other subjects.
All of the findings from the PAD characterization study have been forwarded and shared
directly with the appropriate ASTM subcommittee to ensure that the next revision of the MIST
standard will reflect the recommendations that have been made.
10.2.2 Conclusions Pertaining to the Development of a MIST Manikin
One of the first objectives of the MIST manikin development process was to design a bench-
scale MIST chamber that could be used to quickly expose PADs or potential skin simulant fabrics to
various concentrations of MeS. The NCSU bench-scale MIST chamber was shown to be capable of repeatedly reaching a desired MeS concentration and maintaining the set conditions for an extended period of time. Results from individual fabric exposures were shown to be comparable based on the consistency of background PAD exposures. While the bench-scale chamber was consistent from trial-to-trial, there was a significant discrepancy between the bench-scale exposures and those conducted in the full-scale MIST chamber. These differences are most likely due to the dissimilarity in the flow dynamics directly over the PAD, and should be further investigated.
Fabric exposures in both the bench-scale and full-scale MIST chambers showed that fabrics made of 100% polyester and 100% cotton do not adsorb a significant amount of MeS. The inclusion of 5-10% spandex was shown to have a drastic effect on the uptake rate and capacity for MeS. This finding suggests that fabrics containing high amounts of spandex (greater than 10-20%) could
209 potentially affect the MIST results and therefore should be avoided when evaluating CBRN ensembles on human subjects. If it is deemed necessary to have a skin simulant fabric that would allow a manikin’s nonporous surface to adsorb at a similar rate as human skin, this research has shown that a 95/5 polyester/spandex blended jersey fabric adsorbs MeS at a rate of 3-4 cm/min, which was close to the observed rate of the PADs.
By far the most important phase of the research was the development of the MIST manikin and the investigation into the feasibility of using the manikin in the place of human subjects. One of the main issues with incorporating manikins into the MIST protocol was the overall lack of published data showing comparisons between results between humans and manikins. This void in human- manikin MIST data led to quite a few assumptions about the causes of the assumed differences between manikins and human subjects. Therefore, a key component of this research was to determine the magnitude and level of variability associated with the MIST results from a standard eight human subject test to serve as the baseline for any manikin comparisons. From the human subject testing it can be concluded that there is relatively high level of variability associated with MIST trials. This variability was shown to decrease with the number of test subjects, which justified the requirements of four to eight human subjects stated in the MIST standards. While it was shown that a significant degree of variability does exist in human subject MIST testing, the comparison of the Class 2 and
Class 3 ensembles suggests that the variability has less to do with the subject inside the suit and more to do with the design of the ensemble itself. The Class 2 ensemble, which was designed to provide a higher level of vapor protection than the Class 3 ensemble, was very consistent in many regions of the body across the six human subjects that were tested. These data show that the human subjects could be dressed in the Class 2 ensemble and appropriately seal all of the closure mechanisms each time the ensemble was put on. Consequently, this repeatability led to a lower degree of variation in the
210 results. The Class 3 ensemble, which showed a higher degree of variability from subject to subject in particular body regions, had many more closures and zippers that were more difficult to appropriately seal each time the garment was donned. Therefore, it can be concluded that the variability in MIST results, which is normally viewed as a negative and shortcoming of the test, actually provides extremely useful information about the closure mechanisms that are part of the ensemble.
With the knowledge gained from the human subject testing, the feasibility of incorporating manikins in the MIST protocols could be addressed. First of all, it was shown that an articulated thermal manikin attached to an elliptical walker provides a much more realistic range of motion than the standard manikin walking stand without harming the integrity of the ensemble. Since the appeal of using manikins is tied to belief that they provide less variability than human subjects, the main question that needed to be answered was whether three manikin evaluations would provide both the same magnitude of protection and the same degree of variability as a standard eight human subject test. The human-manikin testing clearly confirmed that three manikin evaluations produce a significantly higher degree of variability than eight human subjects. Thus it is not feasible to only test three manikins instead of eight human subjects. Furthermore, the comparison of the three manikin evaluations to the three replicates of any of the human subjects showed a similar degree of variability.
Since the research showed that the variability in MIST results has more to do with the ensemble design and less to do with the subject inside the ensemble, it is logical to conclude that eight manikin evaluations would provide results consistent in magnitude and degree of variability as eight human subjects.
211 10.3 Proposed Future Research
10.3.1 Future Research on the MIST Methodology
While great strides were made in understanding and improving the standard MIST
methodologies, there continues to be opportunities for future work in this area. It would very
beneficial to further investigate the physical properties of the PADs, in particular the variability in the
thickness and diffusivity of the HDPE film between PAD lots. Also, a further characterization of the
PAD uptake rates at MeS concentrations much lower than previously tested would provide more
information on how the PADs respond in the micro-environment inside the ensemble.
An extremely interesting area of MIST research that could greatly benefit the ensemble designers is in the development of a real-time MeS sensor. A real-time sensor could provide more detailed information on when and where breakthroughs occur during the MIST evaluation. One important note to remember for this area of research is that the purpose of the PADs is to reflect what is actually adsorbed into the skin of the individual whereas a MeS sensor would most likely report a vapor concentration above the skin. A detailed investigation into the body region hazard analysis calculations with the PADs and with the sensors would have to be conducted to correlate the results.
10.3.2 Future Research on MIST Manikin Development and Incorporation
Although the research showed that the original appeal of using manikins to lower the time and cost associated with MIST evaluations was not feasible, there is still future work that could be conducted. First of all, more comparisons between humans and manikins need to be conducted with multiple ensembles of varying levels of protection. These extensive trials would provide even more information on how to interpret the MIST results from either subject type. Also a manikin could be
212 used to investigate the effect of the garment size on the MIST results. If one subject has a 3-XL
ensemble and another subject has a medium ensemble, the difference in air volume available to dilute
the incoming MeS could affect the calculated protection factors. The manikin could provide the
consistent body size to evaluate the differences between garment fit.
An interesting area of manikin research would be the incorporation of the developing physiological models to not only predict chemical protection but to also evaluate the level of heat stress that the individual would endure. The NCSU MIST manikin is capable of heating to desired temperatures, sweating at various rates, and breathing to create the negative pressure inside the respirator. All of these variables can be related to actual human subject data and be programmed to change with the manikin’s work rate. Being able to measure both chemical protection and comfort on an ensemble would be a monumental step forward for MIST evaluations.
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220 APPENDIX
221 APPENDIX A: Body Region Hazard Analysis
A.1 Body Region Hazard Analysis for ASTM F2588 MIST Protocol
A.1.1 Calculation of Local Physiological Protective Dosage Factors
The body region hazard analysis (BRHA) is a set of calculations that determines both localized and systemic protection factors for an ensemble. According to ASTM F 2588 [59], the first step in the BRHA is to determine the uptake rate of the lot of PADs that are being used in the testing.
At least four PADs are to be exposed in the MIST chamber at 100 mg/m3 for 30 ± 5 minutes. After extraction and chromatographic analysis, the total mass of methyl salicylate (MeS) detected on each
PAD is placed into Equation A-1 to calculate the individual PAD uptake rates (cm3/min). The uptake rate for the film (cm/min) can be calculated by incorporating the active sampling surface area of the
PAD into Equation A-1 resulting in Equation A-2. The chamber exposure dosage, Ct, is simply the average chamber concentration multiplied by the time of exposure. The Ct outside of the ensemble is the chamber concentration as determined by the real time analytical measurement multiplied by the exposure duration (Equation A-3). The calculated PAD uptake rates are then averaged to determine the average uptake rate for the lot of PADs. This average uptake rate is then used in Equation A-1 along with the mass of MeS detected on each individual PAD to solve for the Ct inside the ensemble at each PAD location. An example calculation of the Ct(inside) for each PAD location in a Class 2
ensemble is shown in the screen shot of the BRHA program developed at NCSU in Figure A-1.
222
= Equation A-1 푚 푢 3 u - Uptake rate퐶푡 (cm /min)
m - Mass of methyl salicylate adsorbed on PAD (ng)
3 Ct - Chamber exposure dosage (mg.min/m )
= Equation A-2 푚 푢 u - Uptake rate퐴퐶푡 (cm/min)
m - Mass of methyl salicylate adsorbed on PAD (ng) A - Active sampling surface area (cm2)
3 Ct - Chamber exposure dosage (mg.min/m )
( ) = × Equation A-3
표푢푡푠푖푑푒 퐶푡 푀푒푆 퐶표푛푐푒푛푡푟푎푡푖표푛 퐸푥푝표푠푢푟푒 푇푖푚푒 3 Ct(outside) - Chamber exposure dosage outside of ensemble (mg.min/m )
3 MeS Concentration - Concentration measured by FT-IR (mg/m ) Exposure Time - Time (minutes)
223 Local Physiological Protective Dosage Factor Calculation
Date - Customer ID TPACC Research
Subject 1 - Garment ID Cla ss 2 Ense mble
MeS in Extract Total MeS on PAD Ct(inside) PAD Location PAD ID PF PPDF (ng/uL) (ng) (mg.min/m3)
Scalp 1 0.130 138 11.84 549 2197 Forehead 2 0.100 106 9.11 714 2856 Left Ear Upper 3 0.170 180 15.48 420 1680 Left Ear Lower 4 0.160 170 14.57 446 1785 Right Neck Front 5 0.330 350 30.05 216 865 Left Neck Front 6 0.230 244 20.94 310 1242 Nape 7 0.110 117 10.02 649 2596 Armpit 8 0.050 53 4.55 1428 2856 Inner Upper Arm 9 0.080 85 7.28 892 1785 Outer Upper Arm 10 0.050 53 4.55 1428 2856 Left Forearm 11 0.070 74 6.37 1020 2040 Right Forearm 12 0.090 95 8.19 793 1586 Mid Back 13 0.060 64 5.46 1190 4759 Mid Back 14 0.050 53 4.55 1428 5711 Abdomen 15 0.050 53 4.55 1428 5711 Chest 16 0.100 106 9.11 714 2856 Right Butt 17 0.110 117 10.02 649 2596 Lower Back 18 0.040 42 3.64 1785 7139 Groin 19 0.040 42 3.64 1785 7139 Left Crotch 20 0.040 42 3.64 1785 1785 Right Crotch 21 0.050 53 4.55 1428 1428 Left Inner Thigh 22 0.060 64 5.46 1190 4759 Right Inner Thigh 23 0.030 32 2.73 2380 9518 Left Inner Shin 24 0.040 42 3.64 1785 7139 Right Inner Shin 25 0.040 42 3.64 1785 7139 Right Cheek 26 0.120 127 10.93 595 2380 Left Cheek 27 0.120 127 10.93 595 2380 Left Hand 28 0.050 53 4.55 1428 2856 Right Hand 29 0.050 53 4.55 1428 2856 Foot 30 0.040 42 3.64 1785 7139
Body Region of Minimum PPDF Right Neck Front
Minimum PPDF 865
Figure A-1. Calculation of Localized PPDF Values - Screen Shot from Body Region Hazard Analysis Program Developed at NCSU
224 After the Ct(inside) values are calculated, a raw protection factor is generated for each PAD location (i) using the Ct(outside) according to Equation A-4. These raw protection factors are then
weighted to account for the various skin sensitivities in different body regions using Equation A-5 and the site specific onset of symptoms exposure dosage values found in Table A-1. The OSED values as discussed in Section 4.4.2 are based on the ECt10 values for blistering at the given body
regions as a result to exposure to mustard vapor. These local physiological protective dosage factors
(PPDF) are reported for the ensemble’s localized protective performance.
( ) = Equation A-4 ( ) 퐶푡 표푢푡푠푖푑푒 푃퐹푖 퐶푡 푖푛푠푖푑푒 푖 PFi - Protection Factor for individual (i) PAD location
3 Ct(outside) - Chamber exposure dosage outside of ensemble (mg.min/m )
Calculated exposure dosage (mg.min/m3) for individual (i) Ct - (inside)i PAD location
= Equation A.5 25 푖 푂푆퐸퐷 푙표푐푎푙 푃푃퐷퐹푖 푃퐹푖 local PPDFi - Local Physiological Protective Dosage Factor for (i) location
OSEDi - Onset of Symptoms Exposure Dosage (site specific)
PFi - Protection Factor for individual (i) PAD location
225 Table A-1. Site Specific Onset of Symptoms Exposure Dosage (OSED) by PAD Location
Body Region PAD Locations OSED (mg.min/m3)
head/neck 1, 2, 3, 4, 5, 6, 7, 26, 27 100 torso/buttocks 13, 14, 15, 16, 17, 18, 19 100 (excluding perineum) arm/hand 8, 9, 10, 11, 12, 28, 29 50
leg/foot 22, 23, 24, 25, 30 100
perineum 20, 21 25
A.1.2 Calculation of Systemic Physiological Protective Dosage Factor
The local PPDF values only relate to the exposure to locally acting sulfur mustard and do not speak to the ensemble’s ability to protect against nerve agents such as VX which involve a systemic or total body exposure. To calculate the systemic PPDF value for an ensemble the individual PAD locations are converted to represent body regions. The twenty-seven body regions along with their corresponding PAD locations are given in Table A-2. The Ct(inside) values for each PAD in a specific body region are averaged to produce a Ct(inside) for that body region. Then the raw protection factor
for the each body region is calculated according to Equation A-4. A sample calculation for the systemic PPDF value for the previously mentioned Class 2 ensemble is provided in Figure A-2.
226 Table A-2. ED50i Values by PAD and Body Location for Systemic PPDF Calculation
ED for severe effects Body Region (i ) for PADs Mapped to Area of Body Region 50i (VX) for body region BRHA Model Body Region (dz ) i (mg/individual)
Scalp 1, 2 350 0.76 Ears 3, 4 50 0.46 Face, Cheeks, & Neck 5, 6, 26, 27 300 0.48 Chin & Neck 5, 6 200 0.36 Nape 7 100 1.72 Abdomen 16 2858 2.23 Back 13, 14, 18 2540 2.65 Axillae 8 200 2.07 Upper Arm, inner 9 488 2.80 Upper Arm, outer 10 706 6.57 Elbowfold 9, 10, 11, 12 50 2.09 Elbow 9, 10, 11, 12 50 2.25 Forearm, dorsum 11, 12 487 2.80 Forearm, volar 11, 12 706 6.57 Hands, dorsum 28, 29 200 2.91 Hands, palmer 28, 29 200 9.24 Buttocks 17 953 4.26 Groin 15, 19 300 1.22 Crotch 20, 21 200 0.11 Thigh, dorsum 22, 23 2845 6.57 Thigh, plantar 22, 23 1422 4.26 Knee 22, 23, 24, 25 200 7.14 Popliteal Spaces 22, 23, 24, 25 100 2.09 Shins 24, 25 1897 6.57 Calves 24, 25 948 2.80 Feet, dorsum 30 500 6.60 Feet, plantar 30 300 7.14
227 The systemic PPDF value is calculated according to Equation A-6 using the appropriate
values from Table A-2 and the average protection factor for each body region. The top part of
Equation A-6 is simply the area of each body region divided by the ED50 for that specific body
region, and then all of the values are summed up over the total body. The bottom portion of Equation
A-6 is basically the same as the top portion with the exception that each body region is weighted by
the average protection factor for that region. This ratio produces the systemic PPDF value that is
reported as the ensemble’s ability to protect against nerve agent VX.
( 2)
50 ( 푖/ ) 푑푧 푐푚 = � 푖 Equation A-6 퐸퐷 푚푔 푖푛푑푖푣푖푑푢푎푙2 푠푦푠푡푒푚푖푐 ( ) 푃푃퐷퐹 ( / ) 50 푑푧푖 푐푚 � 퐸퐷 푖 푚푔 푖푛푑푖푣푖푑푢푎푙 푃퐹퐵푅
PPDFsystemic - Systemic Physiological Protective Dosage Factor
2 dzi - Area of body region (cm )
Effective Dosage of VX that causes nausea/vomiting in 50% of ED - 50i the exposed population
PFBR - Averaged Protection Factor for specific body region
228 MIST Analysis of Systemic PPDF Values
Date - Customer ID TPACC Research
Subject 1 - Garment ID Cla ss 2 Ense mble
Average Ct(inside) dzi dzi Body Regions for VX for Body Region PF Systemic Calculation 3 ED50i ED50i PFi (mg.min/m ) (cm 2.ind/mg) (cm 2.ind/mg) Scalp 10.47 620.77 460.53 0.74 Ears 15.02 432.66 108.70 0.25 Face, Cheeks, and Neck 18.21 356.94 625.00 1.75 Chin&Neck 25.49 254.96 555.56 2.18 Nape 10.02 648.99 58.14 0.09 Abdomen 9.11 713.89 1281.61 1.80 Back 4.55 1427.77 958.49 0.67 Axillae 4.55 1427.77 96.62 0.07 Upper Arm Medial 7.28 892.36 174.29 0.20 Upper Arm Lateral 4.55 1427.77 107.46 0.08 Elbowfold 6.60 984.67 23.92 0.02 Elbow 6.60 984.67 22.22 0.02 Forearm Extensor 7.28 892.36 173.93 0.19 Forearm Flexor 7.28 892.36 107.46 0.12 Hands dorsum 4.55 1427.77 68.73 0.05 Hands palmar 4.55 1427.77 21.65 0.02 Buttocks 10.02 648.99 223.71 0.34 Groin 4.10 1586.41 245.90 0.16 Scrotum 4.10 1586.41 1818.18 1.15 Thigh anterior 4.10 1586.41 433.03 0.27 Thigh posterior 4.10 1586.41 333.80 0.21 Knee 3.87 1679.73 28.01 0.02 Popliteal Space 3.87 1679.73 47.85 0.03 Shins 3.64 1784.72 288.74 0.16 Calves 3.64 1784.72 338.57 0.19 Feet dorsum 3.64 1784.72 75.76 0.04 Feet plantar 3.64 1784.72 42.02 0.02
∑ 8719.85 10.83
Systemic PPDF 805
Figure A-2. Calculation of Systemic PPDF Values - Screen Shot from Body Region Hazard Analysis Program Developed at NCSU
229 A.2 Body Region Hazard Analysis for Military TOP 10-2-022 Protocol
A.2.1 Calculation of Local and Systemic Protection Factors and Effects
The military standard for MIST testing explains a similar approach to assessing the local and
systemic protection of an ensemble with distinct differences from the ASTM F 2588 protocol. The
first step in the TOP BRHA process is a normalization of the individual mass amounts of MeS on
each PAD so that the results correspond to a standard Ct of 12,000 mg.min/m3 (a 120-minute
exposure at 100 mg/m3). The normalization is calculated using the relationship in Equation A-7, and any normalized values that are below 50 ng (PAD detection limit) are set to 50 ng.
The next step in the BRHA process is to generate average mass amounts for each of the 27 body regions that are represented. A summary of the PADs that correspond to each body region is presented in Table A-3. Since an individual PAD is not located in every body region, some of the regions are estimated from nearby PADs. The specific method to calculating the average mass for each area is also included in Table A-3.
12,000 = Equation A-7
푚푎푠푠푛표푟푚푎푙푖푧푒푑 푚푎푠푠표푟푖푔푖푛푎푙 퐶ℎ푎푙푙푒푛푔푒 퐶푡 massnormalized - Normalized mass on PAD (ng)
massoriginal - Detected mass on individual PAD (ng)
Dosage of MeS (mg.min/m3) measured during the time the challenge Ct - ensemble was tested
230 Table A-3. PADs Mapped to Each Body Region According to TOP 10-2-022
Body Region (i ) for Method to Calculate Average Value for PADs Mapped to Body Region BRHA Model Each Body Region
Scalp P21 Scalp Ears P22 Left Ear Cheeks & Neck P22 Left Ear Chin & Neck P24 Chin Nape P25,PD25 Nape Abdomen P31, P26, P33, PD33 (2*Chest + Armpit + Groin)/4 Back P25, PD25, P30, P32, PD32, P26 (2*(Nape + Middle Back + Buttocks)/3 + Armpit)/3 Axillae P26 Armpit Inner Upper Arm P27 Inner Upper Arm Outer Upper Arm P28 Outer Upper Arm Elbowfold P27, P29 (Inner Upper Arm + Forearm, volar)/2 Elbow P28, P29 (Outer Upper Arm + Forearm, volar)/2 Forearm, dorsum P29 Forearm, volar Forearm, volar P29 Forearm, volar Hands, dorsum P92 Glove Hands, palmer P92 Glove Buttocks P32, PD32 Buttocks Groin P33, PD33, P34 (Groin + Crotch)/2 Crotch P34 Crotch Thigh, dorsum P35 Inner Thigh Thigh, plantar P35 Inner Thigh Knee P35, P36, PD36 (Inner Thigh + Inner Shin)/2 Popliteal Spaces P35, P36, PD36 (Inner Thigh + Inner Shin)/2 Shins P36, PD36 Inner Shin Calves P36, PD36 Inner Shin Feet, dorsum P93 Boot
231 After a normalized mass amount is calculated for each body region, a local protection factor is generated by comparing the mass of MeS adsorbed on a PAD outside of the ensemble to the normalized mass from each body region. A standard value of 200,000 ng is used as the average amount that would be adsorbed on a PAD exposed at 100 mg/m3 for 120 minutes. A better approach would be to expose a set of PADs for each trial at the full challenge dosage and to use the average mass of MeS adsorbed onto those PADs to more accurately reflect the conditions for the particular trial. The calculation of the protection factor is shown by Equation A-8.
200,000 = Equation A-8 ( ) 푛푔 푃퐹퐵표푑푦 푅푒푔푖표푛 푚푎푠푠 퐵표푑푦 푅푒푔푖표푛 PFi - Protection Factor for individual (i) PAD location
3 Ct(outside) - Chamber exposure dosage outside of ensemble (mg.min/m )
Calculated exposure dosage (mg.min/m3) for individual (i) Ct - (inside)i PAD location
After the raw protection factors have been calculated for each body region, an overall systemic protection factor is calculated for the ensemble using the areas and ED50 values from Table
A-2 and Equation A-6 just as with the systemic PPDF calculation in the ASTM F 2588 BRHA. For the TOP BRHA, the systemic protection factor is then multiplied by 25 mg.min/m3, which is the
value that would cause an unprotected individual to experience effects from VX exposure. This value
232 is referred to as the Systemic Minimum Exposure Dosage (MEDsys), and is an estimate of the
ensemble’s protection against agents that act systemically.
To calculate the Minimum Exposure Dosage for sulfur mustard (MEDHD), it is assumed that
the relative sensitivity to VX for each body region is similar to the relative sensitivity to HD.
Essentially, if the armpit is more sensitive to VX than the bottom of the foot, then the armpit would
be equally as more sensitive to HD. At the time of the BRHA development, the forearm was the most
experimented on and most understood body part for VX exposures. Therefore the relative
sensitivities for all body regions are compared to the known values for the forearm. A ratio is created
for each body region using the VX ED50 value for a specific region and the VX ED50 value for the forearm (Table A-2). These ratios are then multiplied by 1,000 mg.min/m3, which is the estimated
dosage of HD that causes severe burns to the forearm. This value is referred to as the local agent Ct50, and is calculated according to Equation A-9. Finally, the local agent Ct50 is multiplied by the protection factor for each body region to result in the clothed effective Ct, a value that estimates the challenge dosage outside of the protective ensemble that would be required to cause chemical burns to the skin of the individual inside the garment (Equation A-10). The MEDHD is the minimum of the
clothed effective Ct values for all body regions. Any of the clothed effective Ct’s or the MEDHD values can be divided by the average MeS concentration during the MIST trial to estimate the time that a subject could remain in the ensemble before receiving a burn to that specific body region.
233
Equation A-1
Vapor challenge to bare skin that would be expected to cause burns Local Agent Ct - 50 (mg.min/m3)
Effective Dosage of VX that causes nausea/vomiting in 50% of the VX ED - 50 region exposed population for a specific body region (mg/individual)
Effective Dosage of VX that causes nausea/vomiting in 50% of the VX ED - 50 forearm exposed population for the forearm (2.8 mg/individual)
Equation A-2
Vapor challenge outside ensemble that would result in burns to the Clothed Effective Ct - individual inside ensemble (mg.min/m3)
Vapor challenge to bare skin that would be expected to cause burns Local Agent Ct - 50 (mg.min/m3)
Local PFBody Region - Local protection factor