IH General Practice II

PO127
IH General Practice II

Wednesday, June 3, 2015, 1:00 PM - 5:00 PM

SR-127-01 Identification of the Electron Dose Threshold for Asbestos Fiber Chemical Analysis by TEM

J. Martin, M. Beauparlant, IRSST, Montreal, QC, Canada; G. L’Espérance, École Polytechnique de Montréal, Montreal, QC, Canada

Objective: Asbestos analysis is still an important part of industrial hygiene analysis. Today a lot of interest is in very fine fibers. Transmission electron microscopy (TEM) ​​is the most powerful technique for counting and identification of asbestos fibres, its high image resolution allows the detection of the smallest fibers and the combination of X-ray energy dispersive spectroscopy analysis (EDS) and selected-area electron diffraction (SAED) leads to an unambiguous identification. While working on the assessment of the current analysis methods, it was observed that the electron dose should be less than a critical value to avoid the loss of certain elements. Exceeding this value leads to a decrease of the X-rays intensity of certain elements relative to others. This is an important part of the identification procedure. The objectives of this research are to define the value of this critical electron dose and determine if this value is unique for all chrysotile fibers, independently of their size.

Methods: All analyses were performed using a 200 kV TEM equipped with a field emission gun (JEOL JEM-2100F) and a Si-Li detector was used for EDS analysis. All chrysotile samples are from UICC Asbestos standards, Chrysotile “A” Rhodesian, on lacey carbon copper TEM grid. All analyses were done at a 100 kV. The electron dose was measured using the phosphorescent screen of the TEM calibrated with a Faraday cage.

Results: The identification of chrysotile thin fibers often relies on elemental composition, and more particularly on the intensity ratio of magnesium and silicon. This research has shown that the characteristics of the electron beam applied on a fiber can significantly influence this ratio. Since magnesium is more weakly bound, it is most affected by radiation damage. Several parameters characterize irradiation: current, beam size and accelerating voltage, they were studied to understand how to avoid this elemental degradation. Many chrysotile fibers of different sizes were tested and in certain cases it was possible to determine the value of the electron dose below which the analysis should be conducted to consider the results stable and reliable.

Conclusions: The optimal parameters of the electron beam in TEM have been identified for the analysis of asbestos thin fibers. A value of electron dose threshold was found for many fibers and can be used to improve the reliability of the current methods.


CS-127-02 Identification of Naturally Occurring Asbestos Work Areas in Coordination with an Exposure Management Program during Vegetation Management Activities

L. O’Heir, San Francisco Public Utilities Commission, San Francisco, CA; B. Drews, Kennedy/Jenks Consultants, San Francisco, CA

Situation/Problem: The San Francisco Public Utilities Commission (SFPUC) performs extensive vegetation management throughout Northern California where naturally occurring asbestos (NOA) is known or suspected. The following steps were followed by SFPUC during their 2010 and 2013 NOA exposure assessments: 1) NOA potential maps were used to identify possible worker exposures and to identify NOA work areas; 2) soil samples were collected to verify NOA map data; 3) employees were trained on NOA work practices; 4) activity based personal air monitoring was performed as part of an Exposure Management Program (EMP); and 5) work was done safely, verified by exposure assessments and field observations.

Resolution: Published NOA map data was compiled into a single geographic information system (GIS) map layer which was superimposed over existing SFPUC GIS infrastructure maps. Bulk samples were collected in work locations to verify NOA mapped areas while personal air samples were collected during routine work tasks.

Results: Fifty-three full shift personal air samples were collected during Phase I (2010) work. Weed whacking produced the highest 8-hour TWA asbestos exposure at 100% of the PEL via TEM analysis. As a result, employee weed whacking practices were amended to reduce exposures. In Phase II (2013), five of nine vegetation management tracts were designated as non-NOA work areas after evaluation of map data in combination with soil sampling results. Twenty-seven (8-hour TWA) and twenty-one (30-minute TWA) personal air samples were collected during Phase II vegetation management activities. Soil discing produced the highest 8-hour TWA asbestos exposure at 43% of the PEL while equipment cleaning produced the highest 30-minute TWA exposure at 11% of the EL (via PCM).

Lessons Learned: Local geology, NOA map data and soil sampling can allow management to distinguish NOA versus non-NOA work locations prior to the start of vegetation management work. Identification of non-NOA work areas can help to reduce operational costs and labor efforts. Activity based air monitoring indicates that a routine activity such as weed whacking can create significant personal exposures from localized emissions. An ongoing EMP is necessary to accommodate wide variations in NOA geological units, work practices and air monitoring results. Key aspects of an EMP should be reflected in task specific Codes of Safe Practice.


SR-127-03 Determinations of Synthetic Musks in Personal Care Products by Solid-Phase Microextraction

W. Tseng, S. Tsai, Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Taiwan

Objective: Emerging environmental pollutants have caused concerned in recent years. For example, a variety of chemical components such as synthetic musks (e.g., galaxolide (HHCB) and tonalide (AHTN)), which have been detected from consumer products, may be released into the environment. To assess the possible health effects, a method for the analysis of HHCB and AHTN in personal care products was developed in this research. The surveillance of the concentrations in food detergent samples in Taiwan was also performed.

Methods: HHCB and AHTN were prepared in mixtures as standard solutions. The samples were first diluted by water and equilibrated for 5 minutes before the extraction. Hence, the extraction was performed at 25°C for 30 minutes with 500 rpm. The 65μm PDMS/DVB fiber was exposed to the headspace over the samples. After adsorption equilibrium has been reached, the SPME fiber was inserted into the injector of the gas chromatography with tandem mass spectrometry for thermal desorption and further analysis.

Results: The SPME procedure coupled with GC/MS/MS analysis for the determinations of HHCB and AHTN in the samples of personal care products was established in this study. No carry-over effect was observed from the thermal desorption of the sample. The linear range of all compounds ranged from 0.005 to 0.05 μg ml-1, and the method detection limits were 0.00015 to 0.00075 μg ml-1. Good linearity and precision were presented. More than 100 food detergents samples in Taiwan were determined for the concentrations of HHCB and AHTN. Health risk associated with the possible exposures were also assessed.

Conclusions: The SPME procedure was applied in this study. Advantages over conventional methods, such as solve-free and time-saving, were reached. The sensitivities of the method for different compounds were low enough to determine the concentrations from personal care products.


SR-127-04 Effects of a Series of Ketone Compounds on Liver Microsomal Aniline Hydroxylase Activity in Mice: Implications for Ketone-Drug Interactions in Exposed Workers

W. Luttrell, Oklahoma Christian University, Edmond, OK

Objective: Exposure to toxic chemicals in the workplace or environment can alter the way the body responds to the administration of therapeutic drugs, other xenobiotics, and endogenous materials. Studies have shown that ketones, such as acetone and pinacolone can produce liver microsomal enzyme enhancement. The purpose of this study was to determine the effect of ketone pretreatment on activity of microsomal aniline hydroxylase activity in the liver of mice. In order to determine if the size of the ketone metabolite influences degree of enzyme enhancement, five ketones of increasing size (acetone, 2-butanone, 2-pentanone, 2-hexanone, and pinacolone) were selected for pretreating mice.

Methods: Groups of male CD-1 mice (n = 6) were pretreated subcutaneously with 90% LDLo (lowest published lethal dose) for each compound and then sacrificed 12 hours later. Liver microsomes were isolated and aniline hydroxylase activity was determined spectrophotometrically. The para-hydroxylation of aniline to para-aminophenol was used to assess the cytochrome P-450-dependent mixed-function oxidase system in liver microsomes. In this assay the para-aminophenol metabolite was chemically converted to a phenol-indophenol complex with an absorption maximum at 630 nm. Enzyme activities were expressed as nmol para-aminophenol formed per minute per milligram protein of tissue. Using a substrate concentration of 10 mM aniline HCl, the velocity of the enzyme reaction was determined in each assay, and was reported as the percent of the control enzyme activity. Using SPSS, statistical differences (p < 0.05) between the values of percent of control enzyme activity from ketone pretreated and control mice were determined.

Results: Aniline hydroxylase activities were increased following pretreatment with all ketone compounds. There was increasing enzyme activity with increasing size of the ketone compound. Lower concentrations of the ketones were found to be enzyme stimulating in vitro, but higher concentrations of the ketones were found to be enzyme inhibiting in vitro.

Conclusions: Pretreatment with ketone compounds of increasing size resulted in increasing liver aniline hydroxylase activity in mice. This implies that workers exposed to these ketone compounds, as well as other common workplace or environmental compounds that have ketone metabolites, may be at risk for ketone-drug interactions due to enhancement of drug-metabolizing enzymes.


SR-127-05 Modern Design of Colorimetric Sensor Devices for Bisphenol A Dust Measurements

A. Rossner, R. Alkasir, S. Andreescu, Clarkson University, Potsdam, NY

Objective: The purpose of our work is to evaluate a new design for a portable paper-based sensing device for the colorimetric detection of Bisphenol A (BPA) in household dust. Bisphenol A (BPA) is found in polycarbonate plastic and epoxy resin and is used in variety a of commercial and consumer products. The leaching of BPA consumer products can result in human exposure via inhalation, ingestion, and dermal routes. As a result, humans have been exposed in their home and work environment to BPA. Several studies have reported detectable levels of total urinary BPA in the majority of individuals in a number of populations, in the United States and other locations around the world. To reduce human exposure we need improved sensing devices to allow for quick, effective and inexpensive screening of our living and working environment.

Methods: A rapid procedure for dust collection is used with a sensitive method for BPA detection, based on the formation of a greenish color, on the test zone of the sensing device. The color results from the formation of a Schiff base compound, quinine-imine, formed by reaction of chitosan with the enzymatic product of tyrosinase o-quinone on paper coated in a layer-by-layer (LbL) assembly approach. The designed system includes a paper-based sensor disk with a diameter of 0.6 cm. as a test zone for BPA detection, and the air-sampling cassette with a diameter of 37 mm as a collection area for household dust. Colorimetric response was concentration dependent with a detection limit of 0.28 µg/g. The color started to appear within the first 60 s and stabilized after 30 min. Replicate samples were run on a Gas Chromatography (GC) as means of validating the colorimetric data. Field sampling was conducted in a series of homes where dust specimens were collected from different homes and a day care center.

Results: Results between the GC and colorimeteric sensor showed a linear regression (R2 = 0.9743) for samples measured by both of the colorimetric and GC methods. In this work, BPA ranged in concentration from 0.05 to 3.87 µg/g in 57 samples of household dust when both methods were used.

Conclusions: While the sample set was relatively small (n=57), the correlation between the colorimetric sensor and GC method was excellent, thus we feel the sensors is promising as a quick, inexpensive means of measuring BPA in settled dust in the home and work environment. 


SR-127-06 Adsorption Characterization of Fabricated Single-Walled Carbon Nanotube (SWNT) Buckypapers for Volatile Organic Compound (VOC) Sampling

C. Lungu, J. Oh, University of Alabama- Birmingham, Birmingham, AL; E. Floyd, OUHSC, Oklahoma City, OK

Objective: To compare adsorption efficiency of single-walled carbon nanotube (SWNT) buckypapers obtained through different fabrication methods to be used as volatile organic compound (VOC) adsorbent.

Methods: Arc discharge SWNTs suspended in surfactants (1% W/V of sodium cholate and sodium dodecyl sulfate) was fabricated into buckypaper. For that, 200 mL (50 mg) of the SWNT solution was suspended (diluted) in 400 mL of acetone for 15 hours, filtered through a polytetrafluoroethylene membrane filter under vacuum. SWNT cake deposited onto the filter was delaminated to obtain a buckypaper (no cleaning process). For cleaning SWNTs, additional steps were involved after SWNT solution was vacuum-filtered. SWNT cake was cleaned with 250 mL of deionized (DI) water and 50 mL of acetone (acetone-cleaned). Alternatively, methanol was used to suspend SWNTs and in the cleaning process as well (methanol-cleaned). The fabricated buckypapers (n=2) were examined for adsorption efficiency in terms of surface area, pore size, and adsorption isotherm. Surface area and pore size were measured using a physisorption analyzer and adsorption isotherms were obtained by repeated indirect injection of liquid toluene into an adsorption chamber until adsorbent was saturated at 23°C.

Results: Buckypapers without cleaning had 43 m2/g Brunauer, Emmett and Teller (BET) surface area (SA) with 15 nm average pore width while buckypapers cleaned with acetone and methanol exhibited 217 and 348 m2/g BET surface areas with 9 and 8 nm average pore widths, respectively. The toluene adsorption capacities were 52, 58, and 69 mg (toluene)/g (buckypaper) for not cleaned, acetone-, and methanol-cleaned buckypapers, respectively.

Conclusions: The cleaning process with DI water and solvents (acetone and methanol) increased BET surface area and decreased the average pore size of the buckypapers. Toluene adsorption capacity increased with increasing surface area of buckypaper. Overall, buckyapers cleaned with DI water and methanol were the most adsorptive. The methanol-cleaned buckypapers will be investigated for desorption efficiency using a photothermal desorption technique which desorbs an analyte by irradiating the sorbent with light, eliminating sample preparation time for analysis.


SR-127-07 Isocyanates: Assessing Field Extraction

D. LaGarde, OSHA, Sandy, UT

Objective: OSHA has operated a National Emphasis Program (NEP) for Isocyanates that began June 2013. The NEP requires OSHA Compliance Officers (CSHOs) to perform a field extraction of air samples and then ship the extracted samples to Salt Lake Technical Center (SLTC) for analysis. Field extraction has been proposed to more effectively derivatize large isocyanate particles, providing higher and more accurate analytical results. Prior to the NEP, OSHA samples were laboratory extracted.

Methods: In an effort to determine the impact of field extraction on compliance work, SLTC staff has been evaluating different aspects of field extraction versus non-field extraction techniques. SLTC performed a stability study to evaluate the necessity of special handling, over-night shipping, and/or cold storage of the field-extracted samples prior to analysis. Sample results were analyzed over the course of the NEP, and statistically evaluated the data against OSHA’s historical sample results. SLTC has coordinated side-by-side samples of non-field extracted and field extracted samples for isocyanates in different occupational processes. To assess the impact of field extraction on sample integrity and sample quality, spiked filters were sent to the field and extracted alongside field samples to assess the impact of field extraction on sample integrity and sample quality.

Results: Sample stability results demonstrated field-extracted samples (glass fiber filters in 3-mL extraction solution), and non-field extracted (dry filters) samples were equivalently stable: recoveries of 100% were obtained for both types of samples after 22-days, at ambient temperature. It has been suggested that field extraction will produce higher results. Sample result data for field extracted samples, show no observed trend in increased sample concentrations over previous years. Results from side-by-side field samples fail to reveal statistical difference between the two sample extraction methods. Quality control spiked samples are used to assess the Sampling and Analytical Error (SAE), a measure of uncertainty in analytical quantitative results. CSHO field extracted isocyanate samples has increased the SAE by over 5%. In addition, a significant percentage of field-extracted samples arriving at SLTC show signs of sample leakage.

Conclusions: Studies performed to date, within the OSHA monitoring program for isocyanate sampling and analysis; show no compelling advantage of field extraction over laboratory extraction.


SR-127-08 Determinations of Bisphenol A in Household Dusts by Microwave-Assisted Solid-Phase Microextraction

C. Chang, C. Chang, S. Tsai, Department of Public Health & Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Taiwan

Objective: Indoor dust has been known as a sink for many semi-volatile organic compounds, such as bisphenol A (BPA). Since indoor environment can protect dust from sunlight, rain and biological degradation, pollutants then could be persistent and accumulated in the residential environment. Up to now, the analysis of BPA in dust is solvent and time consuming. Therefore, the purpose of this research was to develop a method for the determinations of BPA in dusts simultaneously by using microwave assisted headspace solid-phase microextraction (MAE-HS-SPME).

Methods: In this study, commercial vacuum cleaner was used to collect household dusts while particles with diameter smaller than 150μm were filtered out by stainless mesh. After cleaning by the Soxhlet extraction, the sample with known amounts of BPA spiked was then put in a vial for the MAE-HS-SPME extraction followed by the analysis with gas chromatograph and mass spectrometer (GC/MS). Several parameters affecting the SPME extraction efficiency were optimized.

Results: The results showed that the desorption efficiency was 100% when the desorption time was 5 min under 250°C. The best suitable fiber coating was 65μm Polyethylene Glycol (PEG) and the optimum condition of MAE-HS-SPME for extraction of BPA in dust was 20 minutes at 80°C  by the addition of 1 mL water. The linear range for the analysis was 1.25 ~ 125 ng/g dust (r=0.99).

Conclusions: Compared with tradition extraction methods, the MAE-HS-SPME provides a time saving, easy for operation and solvent-free procedure.


CS-127-09 Vacuum Desiccation Applications for the Increase of Polyvinyl Chloride (PVC) Filter Stability

C. Irwin, Galson Laboratories, East Syracuse, NY

Situation/Problem: Polyvinyl Chloride (PVC) filters having a 37-mm diameter and a 5 µm pore width are widely used as industrial hygiene sampling media and are recommended as the media of choice in an array of NIOSH gravimetric methodologies (NIOSH 0500/0600/5000). Precise post sampling quantitation of tared media after analysis depends on the long term stability of the media. The gradual loss of weight in PVC filters was observed over time in both the laboratory setting and in a survey of returned field blanks. Over an 84 day period, laboratory blanks were weighed daily and lost 0.026 ±0.008 mg (n = 15) by day 42. In a separate study of all returned field blanks between January, 1, 2010 and October 21, 2013, filters returned to the lab after 35 days in the field also lost 0.015 ± 0.010 mg (n = 11,475).

Resolution: With both laboratory and field studies indicating a slow loss of weight in un-used PVC filters, the possibility of passive off-gassing of the PVC material in the filter matrix was investigated. A vacuum desiccation study of laboratory media blanks was performed similar in function to the recommended procedure in OSHA PV2121.

Results: After the vacuum desiccation treatment (60°C, 48 hours, -0. 05 MPa), tared PVC filters immediately demonstrated a decline in weight of 0.044 ± 0.028 mg (n=10), and did not regain this weight during the following 42 days of monitoring. Post-treatment analytical accuracy (Standard Deviation*2 of 60 repeated tare weights of the same filter) and coefficient of variance (Standard Deviation*Mean Weight-1 of the same set of 60 repeated filter weights) comparisons between vacuum desiccated and control filters indicate an increase in media stability. The accuracy of 37-mm diameter PVC filters increased from +/- 0.010 mg to +/- 0.008 (95% confidence interval or k=2) and the coefficient of variance decreased from 0.00040 to 0.00030.

Lessons Learned: The vacuum desiccation of PVC sampling media was an effective treatment that increased the reproducibility of initial tare weights in 37-mm diameter 5 µm pore width PVC filter media.


SR-127-10 Effect of Particle Loading on the Performance of Thermal Desorption Tubes

C. Chang, C. Lin, S. Huang, J. Lai, C. Chen, National Taiwan University, Taipei, Taiwan; C. Chang, J. Wu, Chang Jung Christian University, Tainan, Taiwan; J 

Objective: Thermal desorption tubes are commonly used to quantify trace amount of volatile organic compounds in the workplace. These tubes are reused after regeneration. The deposited particles might affect the performance of TDTs. So, this study aimed to characterize the effect of aerosol loading on the performance of TDTs, during both sorption and desorption processes.

Methods: Carbopack X and Tenax TA were used as the sorbents and acetone as the attack agent. The sorbent in the regular package weighed 200 mg. The challenge acetone concentration was set 100 ppm. Flame ionization detector was used for both adsorption and desorption tests to quantify the mass of acetone. For aerosol loading experiment, a constant output atomizer was used to generate submicron aerosol particles. The size distribution was CMD 62.2 nm with GSD of 1.81 and the mass concentration was around 25 mg/m3. A scanning mobility particle sizer was employed to measure the aerosol concentrations and size distributions upstream and downstream of the sorbent. Challenge aerosol flow varied from 0.1 to 0.3 L/min, to study the flow dependency. A pressure transducer was used to monitor the pressure drop during adsorption and desorption.

Results: The 50% breakthrough time of Carbopack X and Tenax TA was 475 s and 235 s, respectively. The collected acetone mass by Carbopack X and Tenax TA was 0.376 and 0.186 mg, respectively. Aerosol penetration of the most penetrating size through 200 mg Carbopack X increased from 9 to 15%, as the aerosol flow increased from 0.1 to 0.3 L/min, indicating that test aerosols entered deeply into the sorbent. The sorption capacity of sorbents could only be reduced by the loading of liquid aerosols. For solid particle loading, the slope of the breakthrough curves became less sharp, meaning shorter 10% breakthrough time and longer 90% breakthrough time. It is inferred that deposited particles acted as a barrier to slow down the organic vapor either going in or coming out the micro-channels of the sorbents.

Conclusions: Liquid aerosols were more poisonous than solid ones because they might form films covering more activated site of sorbents. Aerosol loading deteriorated the sorbent’s performance. The reduction in breakthrough time increased with increasing loaded mass. The glass-wool filter installed upstream of the sorbents would help to protect against aerosol contamination, especially for liquid aerosols.


CS-127-11 Toxicity-Hazard Index and the “Critical Point”

R. Prugh, Chilworth Technology, Inc., Princeton, NJ

Situation/Problem: Recently, the Occupational Safety and Health Administration published Assigned Protection Factors for several types of breathing-protection devices, as part of the OSHA standard in 29 CFR 1910.134(d)(3)(i)(A). Proper use of such devices would allow a person to enter an environment that was at the Maximum Use Concentration of the toxic gas or vapor of interest. Thus, it is essential that the concentration of the gas or vapor be known prior to protected entry into that environment.

Resolution: For many toxic vapors, the “worst-case” equilibrium concentration above a spill of the liquid at a given temperature can be readily calculated from the Antoine vapor-pressure equation - with three coefficients - or, with somewhat greater accuracy, using an equation with five coefficients. Thus, for example, the maximum volumetric concentration of vapor above a spill at “room temperature” can be easily obtained as the ratio of the vapor pressure divided by atmospheric pressure. Then, the type of respirator to protect against that calculation can be determined. Also, the ratio of the maximum equilibrium concentration divided by the OSHA Permissible Exposure Limit could be considered to be a Toxicity-Hazard Index, and this Index would be numerically equal to the required Protection Factor.

Results: However, with novel or complex compounds, the coefficients for a vapor-pressure-versus-temperature equation may not be available. If one set of vapor pressure and temperature values is known, an equation can be derived through use of the “infinite point”, at about 1,625°C and 55,500 psia [about 2.9x106 mmHg].

Lessons Learned: The purpose of this paper is to show how Toxicity-Hazard Indexes can be obtained and how the “infinite point” can be used to estimate room-temperature concentrations of toxic vapors.


SR-127-12 Improvement of Selectivity of the Method for N-Nitrosamine Determination by GC-NPD in Workplace Air

M. Huard, S. Aubin, L René, L. Locas, S. Paradis, IRSST, Montreal, QC, Canada

Objective: The goal of this study was to improve the analytical method for the determination of N-nitrosamines in air by gas chromatography with nitrogen phosphorus detection (GC-NPD). The need was to overcome the problems associated with chromatographic interferences present for field samples. This method helps in evaluating the presence of some carcinogenic N-nitrosamines in the rubber industry.

Methods: For nitrosamine determination, the method used was a sampler adapted by the IRSST and developed by the Institut National de Recherche et de Sécurité (INRS) of France. It consists of a sampling train containing 2 tubes in series. The first tube is a guard to trap amines. The second tube, which contained Florisil®, traps the nitrosamines and only this part is analyzed. The Florisil® is then transferred to a vial, and a liquid-solid extraction is performed with 2 mL of acetone:dichloromethane (98:2) containing an internal standard. The extract is then analyzed by GC-NPD using internal calibration to quantify 8 nitrosamines. Each extract is analyzed twice on two types of configurations simultaneously on the same GC-NPD. A multimode inlet (MMI) with temperature programming is used with a high polarity column for the front NPD detector of the GC. A split/splitless inlet in splitless mode is used with a middle polarity column for the back NPD detector of the GC.

Results: A complete validation was carried out and an LOQ of 0.040 µg per sample was obtained for all 8 nitrosamines, for each column, which corresponds to a concentration in air of 0.06 µg/m³ assuming a sampled volume of 720 L. Some compounds sensitive to thermal degradation can produce undesirable interference if you put them in a hot inlet. Injecting the sample into a cool inlet with the MMI reduced the number of peaks present for some samples and therefore reduced the possibility of interference. Injecting the sample into two different columns (with two different stationary phases) reduced the possibility of false positives, since the probability is really low for an interference to have the same retention time on the two columns.

Conclusions: The method for nitrosamine analysis was therefore improved. The possibility of giving false positives was greatly reduced. Some real sample results will be discussed to demonstrate this conclusion.​