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    DTPQDT02019019640.pdf

    CRAWFORD, STEVEN, Ph.D. Cytotoxicity of Engineered Nanoparticles used in Industrial Processing. 2019 Directed by Dr. James Ryan. 100 pp. Engineered nanoparticles NPs are now heavily used in industrial processing where they are eliminated as waste after use. This waste is a mix of used nanoparticles and process byproducts. While research continues to be done on the toxicity of NPs due to size and composition of pristine material, waste NPs from industrial processes are likely to have modified properties that impact their level of toxicity. These studies investigate this transformation in physicochemical properties that has not been adequately explored by examining waste from relevant high-volume chemical mechanical planarization CMP processes used by the semiconductor industry. New pristine polish slurries and generated waste samples from various key CMP processes are fully characterized for relevant physicochemical properties to determine any transformation of NPs due to processing. Additionally, high throughput in vitro microplate-based assays assess the toxicity, oxidative stress, and mode of cell death for nanoparticles in both pristine and waste slurries to highlight any differences in biological effects. A combination of darkfield microscopy and inductively coupled plasma optical emission spectroscopy ICP-OES indicate cellular uptake of slurry nanoparticles. The results of this study explore the type, magnitude, and biological effect of transformed nanoparticles in CMP waste. The results presented support nanoparticle transformation as an important facet to consider in the risk assessment for new materials. CYTOTOXICITY OF ENGINEERED NANOPARTICLES USED IN INDUSTRIAL PROCESSING by Steven Crawford A Dissertation Submitted to the Faculty of The Graduate School at The University of North Carolina at Greensboro in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Greensboro 2019 Approved by _____________________________ Committee Chair ProQuest Number All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages,these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest Published by ProQuest LLC . Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 13903914 13903914 2019 ii DEDICATION To my wife Kelli and my family for helping me along the way. iii APPROVAL PAGE This dissertation written by Steven Crawford has been approved by the following committee of the Faculty of The Graduate School at the University of North Carolina at Greensboro. Committee Chair______________________________ Committee Members______________________________ ______________________________ ______________________________ Date of Acceptance by Committee 5/15/2019 Date of Final Oral Examination iv TABLE OF CONTENTS Page LIST OF TABLES . vi LIST OF FIGURES vii CHAPTER I. INTRODUCTION .1 I.1 The Effect of Industrial Processing on Engineered Nanoparticles .1 I.2 Hypothesis and Goals .4 II. REVIEW OF THE LITERATURE .6 II.1 Unique Toxicity of Nanoparticles .6 II.2 The Effect of Physichochemical Characteristics .7 II.3 Amorphous Silica Nanoparticles .8 II.4 Nanoparticle Transformation 20 II.5 Exposure and Risk from Chemical Mechanical Planarization 21 III. PHYSICOCHEMICAL CHARACTERIZATION OF NANOPARTICLE SLURRIES BEFORE AND AFTER COMMON CMP PROCESSES 23 III.1 Introduction 23 III.2 Methods25 III.3 Results and Discussion 28 III.4 Conclusions 36 IV. CYTOTOXICITY OF NANOPARTICLE SLURRIES BEFORE AND AFTER COMMON CMP PROCESSES 38 IV.1 Introduction38 IV.2 Methods .38 IV.3 Results and Discussion 42 IV.4 Conclusions60 V. ENVIRONMENTAL IMPACT AND SPECIATION ANALYSIS OF CMP WASTE FOLLOWING GaAs POLISHING 61 V.1 Introduction .61 v V.2 Methods .63 V.3 Results and Discussion66 V.4 Conclusions .75 VI. CONCLUSION AND FUTURE PERSPECTIVES 78 REFERENCES 82 APPENDIX A. CMP PROCESS PARAMETERS 90 vi LIST OF TABLES Page Table 3.1 CMP Processes Used 26 Table 3.2 Slurry and Waste Size Distribution from DLS .33 Table 3.3 Slurry and Waste Zeta Potential Distribution from DLS33 Table 4.1 Cell Viability After 48-Hour Nanoparticle Dose, IC50 Values .46 Table 4.2 Nanoparticle Uptake in RAW 264.7 Macrophages Determined by ICP-OES .52 Table 4.3 Nanoparticle Uptake in A549 Lung Epithelial Cells by ICP-OES .52 Table 4.4 Nanoparticle Uptake in Hep-G2 Liver Epithelial Cells by ICP-OES .53 Table 5.1 Silica Dilution and Arsenic Concentration in Processed Slurry Samples. 68 Table 5.2 AsIII and AsV Levels for CMP Waste Samples .70 vii LIST OF FIGURES Page Figure 1.1 Transformation and Effect of Nanoparticles in the Environment 3 Figure 2.1 Radicals and Silanol Groups that can Exist on an Amorphous Silica Surface .8 Figure 2.2 Representative TEM Images of Round Colloidal Silica NPs A and Chain-like Aggregation of Fumed Silica NPs B. .9 Figure 2.3 Colloidal Silica NPs Internalized in an Endosome .12 Figure 2.4 LDH Release Profile Indicating Membrane Damage for Smaller Silica NPs .14 Figure 2.5 Typical Trends for ROS, MDA, SOD, and GSH-Px that are Concentration Dependent.15 Figure 2.6 Hierarchical Oxidative Stress Model 16 Figure 2.7 The Effect of Silica NPs on G2/M DNA Damage Checkpoint Signaling Pathway Proposed Mechanism .18 Figure 2.8 Hemolysis Caused by Fumed Silica, Not Colloidal Silica NPs19 Figure 2.9 Nanoparticle Transformation and Environmental Fate 21 Figure 3.1 Depiction of CMP Components 24 Figure 3.2 30nm Colloidal Silica Slurry, 63000x TEM Image 31 Figure 3.3 50nm Colloidal Silica Slurry, 50000x TEM Image 31 Figure 3.4 140nm Fumed Silica Slurry, 63000x TEM Image32 Figure 3.5 240nm Alumina Slurry, 63000x TEM Image .32 Figure 3.6 Copper Content and Filter Retention in Waste Slurries .35 viii Figure 4.1 Cell Viability of A549 Lung Epithelial Cells Exposed to 30nm Colloidal Silica above and 140nm Fumed Silica below .47 Figure 4.2 Membrane Damage of A549 Lung Epithelial Cells Exposed to 30nm Colloidal Silica top and 140nm Fumed Silica bottom48 Figure 4.3 Apoptosis Assay Results.49 Figure 4.4 Reactive Oxygen Species Assay Results 50 Figure 4.5 A549 Lung Epithelial Cells, 600x Magnification .54 Figure 4.6 A549 Lung Epithelial Cells Exposed to 30nm SiO2, 600x Magnification 54 Figure 4.7 A549 Lung Epithelial Cells Exposed to 30nm SiO2, 600x Magnification 55 Figure 4.8 A549 Lung Epithelial Cells Exposed to 140nm fumed SiO2, 600x Magnification 55 Figure 4.9 A549 Lung Epithelial Cells Exposed to 200nm CeO2, 600x Magnification 56 Figure 4.10 A549 Lung Epithelial Cells Exposed to 240nm Al2O3, 600x Magnification 56 Figure 4.11 HepG2 Liver Epithelial Cells, 600x Magnification 57 Figure 4.12 HepG2 Liver Epithelial Cells Exposed to 240nm Al2O3, 600x Magnification 57 Figure 4.13 RAW 264.7 Macrophage Cells, 600x Magnification .58 Figure 4.14 RAW 264.7 Macrophage Cells Exposed to 30nm SiO2, 600x Magnification 58 Figure 4.15 RAW 264.7 Macrophage Cells Exposed to 140nm fumed SiO2, 600x Magnification 59 Figure 5.1 Size Distribution of Slurry NPs in Stock Slurry, Wet Etch Bath, and CMP Waste. 66 ix Figure 5.2 TEM Image of Slurry NPs a Before and b after CMP, 80,000x Magnification. 68 Figure 5.3 Arsenic Species Ratio for CMP Waste and Wet Etch Samples 71 Figure 5.4 Arsenic Speciation Following pH Adjustment of Dilute 9.0 psi CMP Waste Sample. 71 Figure 5.5 Viability a and Membrane Damage b of A549 Lung Epithelial Cells Exposed to Stock Slurry Containing Silica NPs and Wastewater from CMP of GaAs .74 1 CHAPTER I INTRODUCTION Engineered nanoparticles NPs are synthesized and used for a wide variety of industrial and consumer products. Metals, metal oxide, and polymer nanoparticles can be found in products that come into direct contact with the general public, while others may enter the environment during synthesis or use. The global nanoparticle market has experienced growth over the past two decades in diverse fields such as aerospace, automotive, catalysts, coatings, paints, pigments, composites, cosmetics, electronics and optics, energy, filtration and purification, plastics, textiles and many others. To this end, approximately 300,000 metric tons of engineered nanomaterials were used just in 2010. While occupational exposure of researchers and engineers represent the first group at risk, most of these materials end up in landfills, the soil, or water. The growing prevalence and use of these various nanomaterials increase the average person’s likelihood to be exposed to them over the course of one’s life. I.1 The Effect of Industrial Processing on Engineered Nanoparticles In the growing global market for engineered nanoparticles, electronic applications in the semiconductor industry make up the lion’s share at well over 1 billion annually. For the last decade, the largest segment has been polishing slurries for chemical- 2 mechanical planarization CMP. Silicon oxide, cerium IV oxide and aluminum oxide nanoparticles are among those most commonly used by modern processing techniques. [1] The CMP process uses massive quantities of these nanoparticles in a slurry form to polish and planarize wafers via some ratio of both chemical interaction and/or tribo- mechanical removal of material. [38] As material is removed from wafers, it is mixed with the used slurry and rinse water and then discharged after varying degrees of treatment or reclamation depending on the region. [7,37] Tens of liters of waste slurry is generated each wafer polish cycle, and 40 of all water used in the manufacture of integrated circuits is consumed by CMP. [2, 9, 22] Huge quantities of nanoparticles are used in slurries so wastewater from the CMP process can pose a health hazard by the pollution of water systems and soil with these engineered nanoparticles. [25, 47, 55] In addition to any intrinsic toxicity of the bulk materials, nanoparticles can be more toxic due to their high surface area and ability to penetrate biological membranes. [15, 33, 34] Due to the mixed chemical and mechanical nature of the CMP process, the slurry nanoparticles may also become impregnated or bound to lesser amounts of the removed substrate material, further complicating and potentially increasing the toxicity of the mixture. [3] Although the toxicity profile of many of the pure materials used in the process have been investigated, the effect of mixing potentially transformed nanoparticles with removed substrate material is poorly understood and has not been researched adequately. [3, 5, 6, 11, 14, 19, 20, 23, 24, 31, 32, 36] 3 Figure 1.1 Transformation and Effect of Nanoparticles in the Environment. [53] Previous studies and existing literature pertaining to the toxicity of CMP waste have not adequately characterized physicochemical properties to assess any transformation or modification to the engineered nanoparticles resulting from the process. [29] The toxicity profiles of nanomaterials can vary tremendously based on size, surface area, surface chemistry, and many other properties, even with nanoparticles of similar chemical composition. [13, 15, 16, 17] Understanding the hazards of only the newly manufactured, pristine nanomaterials is not sufficient for the regulation of transformed waste that is discharged in a modified form. Waste needs to be investigated for toxicity in comparison to its original pristine form to determine the biological effect of physicochemical changes. To make sense of the complicated landscape of nanoparticle health and safety, any changes in toxicity need to be tracked, studied, and attributed to the physicochemical property changes that can result from industrial processing. [29] This information will enable the development of new methods for evaluating the health and safety impacts of new processes or materials with an emphasis on green engineering. 4 I.2 Hypothesis and Goals This dissertation details the work undertaken to explore the science behind process-related nanoparticle transformation and its effect on mammalian cells. The hypothesis that motivated this research is that in industrial processes that use NPs such as CMP, waste NPs are likely to modify/transform physicochemical properties of those NPs, which may result in altered toxicity and associated hazards. Specifically, this research sought to establish that A CMP slurry NPs will be transformed by the CMP process and B those transformed NPs will cause a discernible difference in cellular toxicity. To test the hypothesis, specific aims and objectives were established 1 Specific Aim Assess the transformation of nanoparticles in common slurry types from a representative group of CMP polish processes. a. Objective 1.1 Generate and characterize physicochemical properties of pristine and waste slurry samples to assess transformation of NPs. 2 Specific Aim Compare pre- and post-process nanoparticles for effect on cell viability, mode of cell death, and oxidative stress in lung, liver, and macrophage cells. a. Objective 2.1 Determine dose-response for cell viability. b. Objective 2.2 Determine mode of cell death and ROS generation level 3 Specific Aim Assess any differences in the amount of nanoparticle uptake and localization for transformed nanoparticles versus pristine. a. Objective 3.1 Determination of total nanoparticle uptake by ICP-OES 5 b. Objective 3.2 Darkfield imaging to determine localization In the completion of these aims and objectives, relevant off-the-shelf CMP slurries and common in vitro immortalized cell lines were used. All process and slurry combinations were characterized and used for toxicity studies, regardless of detectable transformation in properties. Additional objectives specific to one process, GaAs polishing, were also added and explored. Due to the unique results of this process, it is explored on its own in Chapter V. 6 CHAPTER II REVIEW OF THE LITERATURE II.1 Unique Toxicity of Nanoparticles Nanoparticles and other nanomaterials have distinct toxic effects from bulk material or molecular forms. Over the past two decades, research has shown that the unique physicochemical characteristics of nanomaterials are the reason for their unique toxicology. Increased surface area with small size compared to other particulate toxicants, unique surface groups and structure, coatings, and aggregating properties have all been shown to play a role in toxicity. Regardless of chemistry, nanomaterials can play a role in the biological machinery at cellular and subcellular levels. At the lowest end of the nanoscale range, many materials diffuse easily through all membranes and

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