A Review of Selected Engineered Nanoparticles in the Atmosphere: Sources,Transformations, and Techniques for Sampling and Analysis

Abstract

A state-of-the-science review was undertaken to identify and assess sampling and analysis methods to detect and quantify selected nanomaterials (NMs) in the ambient atmosphere. The review is restricted to five types of NMs of interest to the Office of Research and Development Nanomaterial Research Strategy (US Environmental Protection Agency): cerium oxide, titanium dioxide, carbon nanostructures (carbon nanotubes and fullerenes), zero-valent iron, and silver nanoparticles. One purpose was determining the extent to which present-day ultrafine sampling and analysis methods may be sufficient for identifying and possibly quantifying engineered NMs (ENMs) in ambient air. Conventional sampling methods for ultrafines appear to require modifications. For cerium and titanium, background levels from natural sources make measurement of ENMs difficult to quantify. In cases where field studies have been performed, identification from bulk analysis samples have been made. Further development of methods is needed to identify these NMs, especially in specific size fractions of ambient aerosols.     

Manufacture and use of nanomaterials: current status in the UK and global trends

This paper provides an overview of the production and use of nanomaterials (NMs), particularly in the UK. Currently, relatively few companies in the UK are identifiable as NM manufacturers, the main emphasis being the bulk markets in metals and metal oxides, and some niche markets such as carbon nanotubes and quantum dots. NM manufacturing in the UK does not reflect the global emphasis on fullerenes, nanotubes and fibres. Some assumptions have been made about the types of NM that are likely to be imported into the UK, which currently include fullerenes, modified fullerenes and other carbon-based NMs including nanotubes. Many university departments, spin-offs and private companies have developed processes for the manufacture of NMs but may only be producing small quantities for research and development (R&D) purposes. However, some have the potential to scale up to produce large quantities. The nanotechnology industry in the UK has strong R&D backup from universities and related institutions. This review has covered R&D trends at such institutions, and appropriate information has been added to a searchable database. While several companies are including NMs in their products, only a few (e.g. manufacturers of paints, coatings, cosmetics, catalysts, polymer composites) are using nanoparticles (NPs) in any significant quantities. However, this situation is likely to change rapidly. There is a need to collect more information about exposure to NPs in both manufacturing and user scenarios. As the market grows, and as manufacturers switch from the micro- to the nanoscale, the potential for exposure will increase. More research is required to quantify any risks to workers and consumers.     

Analysis of engineered nanomaterials in complex matrices (environment and biota): general considerations and conceptual case studies

Advances in the study of the environmental fate, transport, and ecotoxicological effects of engineered nanomaterials (ENMs) have been hampered by a lack of adequate techniques for the detection and quantification of ENMs at environmentally relevant concentrations in complex media. Analysis of ENMs differs from traditional chemical analysis because both chemical and physical forms must be considered. Because ENMs are present as colloidal systems, their physicochemical properties are dependent on their surroundings. Therefore, the simple act of trying to isolate, observe, and quantify ENMs may change their physicochemical properties, making analysis extremely susceptible to artifacts. Many analytical techniques applied in materials science and other chemical/biological/physical disciplines may be applied to ENM analysis as well; however, environmental and biological studies may require that methods be adapted to work at low concentrations in complex matrices. The most pressing research needs are the development of techniques for extraction, cleanup, separation, and sample storage that introduce minimal artifacts to increase the speed, sensitivity, and specificity of analytical techniques, as well as the development of techniques that can differentiate between abundant, naturally occurring particles, and manufactured nanoparticles.     

Engineered nanomaterials: exposures, hazards, and risk prevention

Nanotechnology presents the possibility of revolutionizing many aspects of our lives. People in many settings (academic, small and large industrial, and the general public in industrialized nations) are either developing or using engineered nanomaterials (ENMs) or ENM-containing products. However, our understanding of the occupational, health and safety aspects of ENMs is still in its formative stage. A survey of the literature indicates the available information is incomplete, many of the early findings have not been independently verified, and some may have been over-interpreted. This review describes ENMs briefly, their application, the ENM workforce, the major routes of human exposure, some examples of uptake and adverse effects, what little has been reported on occupational exposure assessment, and approaches to minimize exposure and health hazards. These latter approaches include engineering controls such as fume hoods and personal protective equipment. Results showing the effectiveness - or lack thereof - of some of these controls are also included. This review is presented in the context of the Risk Assessment/Risk Management framework, as a paradigm to systematically work through issues regarding human health hazards of ENMs. Examples are discussed of current knowledge of nanoscale materials for each component of the Risk Assessment/Risk Management framework. Given the notable lack of information, current recommendations to minimize exposure and hazards are largely based on common sense, knowledge by analogy to ultrafine material toxicity, and general health and safety recommendations. This review may serve as an overview for health and safety personnel, management, and ENM workers to establish and maintain a safe work environment. Small start-up companies and research institutions with limited personnel or expertise in nanotechnology health and safety issues may find this review particularly useful.     

Ecotoxicity test methods for engineered nanomaterials: practical experiences and recommendations from the bench

Ecotoxicology research is using many methods for engineered nanomaterials (ENMs), but the collective experience from researchers has not been documented. This paper reports the practical issues for working with ENMs and suggests nano-specific modifications to protocols. The review considers generic practical issues, as well as specific issues for aquatic tests, marine grazers, soil organisms, and bioaccumulation studies. Current procedures for cleaning glassware are adequate, but electrodes are problematic. The maintenance of exposure concentration is challenging, but can be achieved with some ENMs. The need to characterize the media during experiments is identified, but rapid analytical methods are not available to do this. The use of sonication and natural/synthetic dispersants are discussed. Nano-specific biological endpoints may be developed for a tiered monitoring scheme to diagnose ENM exposure or effect. A case study of the algal growth test highlights many small deviations in current regulatory test protocols that are allowed (shaking, lighting, mixing methods), but these should be standardized for ENMs. Invertebrate (Daphnia) tests should account for mechanical toxicity of ENMs. Fish tests should consider semistatic exposure to minimize wastewater and animal husbandry. The inclusion of a benthic test is recommended for the base set of ecotoxicity tests with ENMs. The sensitivity of soil tests needs to be increased for ENMs and shortened for logistics reasons; improvements include using Caenorhabditis elegans, aquatic media, and metabolism endpoints in the plant growth tests. The existing bioaccumulation tests are conceptually flawed and require considerable modification, or a new test, to work for ENMs. Overall, most methodologies need some amendments, and recommendations are made to assist researchers.     

Critical review of methods for sampling,analysis, and monitoring of vapor-phase toluene diisocyanate

This article is a critical review of some of the methods that have been used for the sampling, analysis, and monitoring of vapor-phase toluene diisocyanate (TDI). Only some of the methods that have received relatively widespread application have been addressed in this article. This review includes a "tutorial style" discussion of basic definitions and basic principles and procedures of quality control and metrology for sampling, analysis, and monitoring. One critical issue that is addressed: Are there methods and monitoring instruments available that are capable of sampling and analyzing or monitoring TDI vapor with sufficient speed and sensitivity to satisfy the requirements of the current ACGIH threshold limit values (TLVs) for TDI vapor?     

Determination of carbon disulfide at the workplace by sampling on charcoal tubes--problems and solutions

The aim of the study was to check the reliability and comparability of different analytical methods for ambient monitoring of carbon disulfide (CS2). A stationary sampling system, consisting of a charcoal sampling tube and pump, and two personal sampling systems, consisting of a charcoal sampling tube and a portable pump and of a diffusive charcoal sampler have been compared. The limits of quantification, within-series precision, between-series precision, recovery, and comparability of the methods were investigated. For passive sampling the recovery was determined by three different techniques. For a sampling time of 6 hours the limit of quantification was 0.2 ppm for the personal sampling and 0.01 ppm for the stationary sampling system. The within-series precision was between 5 and 8%. For personal sampling the between-series precision was between 9 and 12% using a passive sampler. The recovery ranged between 45 and 85% depending on the quotient of eluent volume and charcoal mass. The comparison of the two personal sampling methods in a field study using linear regression demonstrated with statistical certainty an excellent concordance of the methods. The study shows that particularly the method for determining air levels of carbon disulfide by passive sampling is associated with high systematic errors. If these errors are unknown or ignored they will result in highly underestimated exposure data for carbon disulfide.     

Statistical comparison of diesel particulate matter measurement methods

Four methods are used to quantify diesel particulate matter (DPM) in the mine environment: respirable combustible dust sampling (RCD), size selective sampling with gravimetric analysis (SSG), respirable dust sampling with elemental carbon (EC) analysis, and respirable dust sampling with total carbon (TC) analysis. The authors assembled data from three underground mine studies to statistically compare these methods. The sampling protocol used in each study was similar. For all the four methods, samples were collected in triplicate at three locations-upwind and downwind of the diesel scoop and on the scoop. The methods were compared with respect to their precision, selectivity, sensitivity, and LOD, as well as their limitations in measuring DPM concentrations. This constitutes a meta-analysis of the available data and provides information over a broader range of mining conditions and DPM concentrations than any of the individual studies. The weighing imprecision for the SSG method is almost twice that for the RCD technique. The imprecision of the EC and TC methods are a function of the mass loading, and EC has a lower imprecision than TC. The EC method was used as the reference "gold standard" against which the other methods were evaluated. The RCD, SSG, and TC methods exhibited substantial levels of interference, leading to much higher minimum concentrations that can be measured by these methods. Of the three, the SSG method has the highest level of interference, primarily from nondiesel material that is collected in the <0.8 microm size range.     

Passive dosimeters for nitrogen dioxide in personal/indoor air sampling: a review

Accurate measurement of nitrogen dioxide concentrations in both outdoor and indoor environments, including personal exposures, is a fundamental step for linking atmospheric nitrogen dioxide levels to potential health and ecological effects. The measurement has been conducted generally in two ways: active (pumped) sampling and passive (diffusive) sampling. Diffusion samplers, initially developed and used for workplace air monitoring, have been found to be useful and cost-effective alternatives to conventional pumped samplers for monitoring ambient, indoor and personal exposures at the lower concentrations found in environmental settings. Since the 1970s, passive samplers have been deployed for ambient air monitoring in urban and rural sites, and to determine personal and indoor exposure to NO2. This article reviews the development of NO2 passive samplers, the sampling characteristics of passive samplers currently available, and their application in ambient and indoor air monitoring and personal exposure studies. The limitations and advantages of the various passive sampler geometries (i.e., tube, badge, and radial type) are also discussed. This review provides researchers and risk assessors with practical information about NO2 passive samplers, especially useful when designing field sampling strategies for exposure and indoor/outdoor air sampling.     

Sunlight exposure assessment: can we accurately assess vitamin D exposure from sunlight questionnaires?

The purpose of this review is to summarize the peer-reviewed literature in relation to sunlight exposure assessment and the validity of using sunlight exposure questionnaires to quantify vitamin D status. There is greater variability in personal ultraviolet (UV) light exposure as the result of personal behavior than as the result of ambient UV light exposure. Although statistically significant, the correlation coefficients for the relation between personal report of sun exposure and ambient UV light measured by dosimetry (assessment of radiation dose) are relatively low. Moreover, the few studies to assess the relation between sunlight measures and serum 25-hydroxyvitamin D show low correlations. These low correlations may not be surprising given that personal factors like melanin content in skin and age also influence cutaneous synthesis of vitamin D. In summary, sunlight exposure questionnaires currently provide imprecise estimates of vitamin D status. Research should be directed to develop more objective, nonintrusive, and economical measures of sunlight exposure to quantify personal vitamin D status.     

Associations between short-term changes in nitrogen dioxide and mortality in Canadian cities

The association between daily variations in ambient concentrations of nitrogen dioxide (NO2) and mortality was examined in 12 of Canada's largest cities, using a 19-yr time-series analysis (from 1981-1999). The authors employed parametric statistical methods that are not subject to the recently discovered convergence and error estimation problems of generalized additive models. An increase in the 3-d moving average of NO2 concentrations equivalent to the population-weighted study mean of 22.4 ppb was associated with a 2.25% (t = 4.45) increase in the daily nonaccidental mortality rate and was insensitive to adjustment for ozone, sulfur dioxide, carbon monoxide, coefficient of haze, size-fractionated particulate mass, and the sulfate ion measured on an every-6th-day sampling schedule. The 3-d moving average of NO2 was sensitive to adjustment for fine particulate matter measured daily during the 1998-2000 time period.     

Characterization of cerium oxide nanoparticles-part 2: nonsize measurements

Part 1 (see companion paper) of the present study discussed the application of a multimethod approach in characterizing the size of cerium oxide nanoparticles (NPs). However, other properties less routinely investigated, such as shape and morphology, structure, chemical composition, and surface properties, are likely to play an important role in determining the behavior, reactivity, and potential toxicity of these NPs. The present study describes the measurement of the aforementioned physicochemical properties of NPs (applied also to nanomaterials [NMs]) compared with micrometer particles (MPs). The authors use a wide range of techniques, including high resolution-transmission electron microscopy, scanning electron microscopy, atomic force microscopy, X-ray diffraction, X-ray energy dispersive spectroscopy, electron energy loss spectroscopy, X-ray photoelectron spectroscopy, and electrophoresis, and compare these techniques, their advantages, and their limitations, along with recommendations about how best to approach NM characterization, using an application to commercial cerium oxide NPs and MPs. Results show that both cerium oxide NPs and MPs are formed of single polyhedron or truncated polyhedron crystals. Cerium oxide NPs contain a mixture of Ce(3+) and Ce(4+) cations, whereas the MPs contain mainly Ce(4+) , which is potentially important in understanding the toxicity of cerium oxide NPs. The isoelectric point of cerium oxide NPs was approximately pH 8, which explains their propensity to aggregate in aqueous media (see companion paper).     

On the release of asbestos fibers from weathered and corroded asbestos cement products

The controversy on whether weathered and corroded asbestos cement products are emitting biologically significant asbestos fiber concentrations in ambient air has not been resolved. Nor is it known if the weathered and corroded asbestos cement products release asbestos fibers which have the same carcinogenic potency as "standard" chrysotile. The purpose of this research project was to develop a method for sampling and measuring asbestos fiber emissions from solid planar surfaces (i.e., roofs and facades) consisting of asbestos cement products and to develop methods for studying the physical and chemical changes and the carcinogenic potency of the emitted fibers. Using this method asbestos fiber emissions in ambient air have been measured in the FRG during 1984/1986. The emissions of asbestos fibers longer than 5 microns were in the range 10(6) to 10(8) fibers/m2.hr. The ambient air concentrations of these asbestos fibers were for the most part less than 10(3) fibers/m3. It was shown that the emitted asbestos fibers were chemically changed and it was shown with animal experiments that their carcinogenic potency did not differ from the carcinogenicity of "standard" chrysotile fibers.     

Breast feeding practices in urban and rural health centres: impact of baby friendly hospital initiative in Ile-Ife, Nigeria

The present study was carried out to assess the impact of the Baby Friendly Hospital Initiative (BFHI) on breast feeding practices. The variables used to evaluate the BFHI centre included time of initiation of breastfeeding (BF) after delivery, intended duration of BF, adoption of exclusive breastfeeding (EBF) in relation to educational status, location of health centre, and knowledge and practice of positioning the child at the breast. Observation, questionnaire and interview techniques were employed to collect the data among 217 and 214 nursing mothers (NMs) from Urban and Rural Health Centres. Of the 228 NMs who initiated BF within 30 minutes after delivery, 140 (61%) were from a designated BFHI Centre and 88 (39%) were from an undesignated BFHI rural health centre. There was a significant relationship between the practice of EBF and designation of BFHI centre (P = 0.0001). The higher the level of education of the NMs, the higher the present of NMs subscribing to EBF (P = 0.0001). The present study has also confirmed that EBF which was once considered to be less than 3%, has increased significantly to 61%. The success was not unconnected with the inauguration of BFHI. The BFHI is recording huge success in the urban area but the impact is still to be felt in the rural areas.     

Interlaboratory Evaluation of Rodent Pulmonary Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium

Background: Engineered nanomaterials (ENMs) have potential benefits, but they also present safety concerns for human health. Interlaboratory studies in rodents using standardized protocols are needed to assess ENM toxicity.Methods: Four laboratories evaluated lung responses in C57BL/6 mice to ENMs delivered by oropharyngeal aspiration (OPA), and three labs evaluated Sprague-Dawley (SD) or Fisher 344 (F344) rats following intratracheal instillation (IT). ENMs tested included three forms of titanium dioxide (TiO₂) [anatase/rutile spheres (TiO₂-P25), anatase spheres (TiO₂-A), and anatase nanobelts (TiO₂-NBs)] and three forms of multiwalled carbon nanotubes (MWCNTs) [original (O), purified (P), and carboxylic acid “functionalized” (F)]. One day after treatment, bronchoalveolar lavage fluid was collected to determine differential cell counts, lactate dehydrogenase (LDH), and protein. Lungs were fixed for histopathology. Responses were also examined at 7 days (TiO₂ forms) and 21 days (MWCNTs) after treatment.Results: TiO₂-A, TiO₂-P25, and TiO₂-NB caused significant neutrophilia in mice at 1 day in three of four labs. TiO₂-NB caused neutrophilia in rats at 1 day in two of three labs, and TiO₂-P25 and TiO₂-A had no significant effect in any of the labs. Inflammation induced by TiO₂ in mice and rats resolved by day 7. All MWCNT types caused neutrophilia at 1 day in three of four mouse labs and in all rat labs. Three of four labs observed similar histopathology to O-MWCNTs and TiO₂-NBs in mice.Conclusions: ENMs produced similar patterns of neutrophilia and pathology in rats and mice. Although interlaboratory variability was found in the degree of neutrophilia caused by the three types of TiO₂ nanoparticles, similar findings of relative potency for the three types of MWCNTs were found across all laboratories, thus providing greater confidence in these interlaboratory comparisons.     

Effects of burnrate, wood species, altitude, and stove type on woodstove emissions

During the winter of 1986-87, the U.S. Environmental Protection Agency (EPA) conducted an emission measurement program in Boise, ID, as part of the Integrated Air Cancer Project (IACP). This program was designed to identify the potential mutagenic impact of residential wood burning on ambient and indoor air. One facet of this field sampling effort involved obtaining emission samples from chimneys serving wood burning appliances in Boise. As a companion to the field source sampling, a parallel project was undertaken in an instrumented woodstove test laboratory to quantify woodstove emissions during operations typical of Boise usage. Two woodstoves were operated in a test laboratory over a range of burnrates, burning either eastern oak or white pine from the Boise, ID, area. A conventional stove, manufactured in the Boise area, was tested at altitudes of 90 and 825 m. A catalytic stove was tested only at the high altitude facility. All emission tests were started with kindling a fire in a cold stove using black and white newsprint. Emissions were collected using the wood stove dilution sampling system (WSDSS) for a continuous period of about 8 hours, encompassing start-up and several wood additions. The results showed wide variability probably due primarily to the difficulty in duplicating conditions during start-up. Total WSDSS emissions showed the expected inverse correlations with burnrate for the conventional stove and nearly flat for the catalytic stove. While there appeared to be little or no correlation of total WSDSS emissions with altitude, the sum of the 16 polynuclear aromatic hydrocarbons (PAHs) quantified showed an inverse correlation with altitude: higher PAH emissions at the lower altitude.     

Endotoxin,cat, and house dust mite allergens in electrostatic cloths and bedroom dust

Environmental exposure to endotoxin, Fel d I (cat) allergen and Der p I (house dust mite) allergen have been associated with asthma symptoms and have been measured in the environment using various sampling methods, including the electrostatic dust collector. The objectives of this study were to investigate whether levels of endotoxin and allergens were detectable in electrostatic dust collectors and to examine the correlation of allergen and endotoxin levels between electrostatic dust collectors and vacuum sampling methods (floor dust and mattress dust). Electrostatic cloths, bedroom floor dust and mattress dust samples from a subset of 60 homes were randomly selected from the Health of Occupants of Mouldy Homes study for allergen and endotoxin analysis. Fel d I and Der p I allergens were analyzed by double monoclonal antibody ELISA and endotoxin by the kinetic Limulus amoebocyte lysate assay. An enhanced ELISA method was used to analyze Der p I in the electrostatic cloths. Endotoxin was detected in all samples, however Fel d I and Der p I were not detected in all electrostatic dust collector samples (detection in 53% and 15% of cloths respectively). No correlations were found between cloth and dust samples for endotoxin or Der p I, but moderate-to-strong correlations were found between all three sampling methods for Fel d I (r_s = 0.612–0.715, p < 0.001). Poor correlation was found between floor dust and mattress dust samples for Der p I (r_s = 0.256, p = 0.048). Electrostatic dust collectors may provide a way to measure airborne dust and allergen. Given the moderate-to-low correlations with vacuum dust sampling, this may present a unique measurement system which, when collected alongside traditional vacuum dust sampling, could provide additional exposure measures. Further studies are required to correlate endotoxin and allergen levels measured by electrostatic dust collector with air sampling and to explore the relationships between these bioaerosols, environmental factors and asthma.     

New cerium(III) complexes of coumarins - synthesis, characterization and cytotoxicity evaluation

Complexes of cerium(III) with bis(4-hydroxy-2-oxo-2H-chromen-3-yl)-pyridin-2-yl-methane, bis(4-hydroxy-2-oxo-2H-chromen-3-yl)-pyridin-3-yl-methane and bis(4-hydroxy-2-oxo-2H-chromen-3-yl)-pyridin-4-yl-methane were synthesized by reaction of cerium(III) salt and the ligands, in amounts equal to metal-ligand molar ratio of 1:2. The cerium(III) complexes with bis-coumarins were characterized by different physicochemical methods - elemental analysis, IR-, Raman-, (1)H NMR- and (13)C NMR-spectroscopy and mass-spectral data. The spectral data of cerium(III) complexes were interpreted on the basis of comparison with the spectra of the free ligands. This analysis showed that in the Ce(III) complexes the ligands coordinated to the metal ion through both deprotonated hydroxyl groups. On the basis of the nu(CO) red shift observed, participation of the carbonyl groups in the coordination to the metal ion was also suggested. Cytotoxic screening by MTT assay was carried out. In the present study we performed comparative evaluation of the cytotoxic effects of the three newly synthesized cerium complexes against the acute myeloid leukemia derived HL-60 and the chronic myeloid leukemia (CML)-derived BV-173. In addition the cytotoxic effects of Ce(III) complex with bis(4-hydroxy-2-oxo-2H-chromen-3-yl)-pyridin-2-yl-methane were evaluated on the SKW-3 cells. In order to elucidate some of the mechanistic aspects of the observed cytotoxic effects we evaluated the ability of this complex to trigger programmed cell death (apoptosis by means of agarose gel electrophoretic analysis of DNA, isolated from the cytosolic fraction of treated SKW-3 cells). In addition, microscopic morphological evaluation of the treated cells was carried out in order to establish morphological features indicative of programmed cell death.     

Associations between air pollution and mortality in Phoenix, 1995-1997

We evaluated the association between mortality outcomes in elderly individuals and particulate matter (PM) of varying aerodynamic diameters (in micrometers) [PM(10), PM(2.5), and PM(CF )(PM(10) minus PM(2.5))], and selected particulate and gaseous phase pollutants in Phoenix, Arizona, using 3 years of daily data (1995-1997). Although source apportionment and epidemiologic methods have been previously combined to investigate the effects of air pollution on mortality, this is the first study to use detailed PM composition data in a time-series analysis of mortality. Phoenix is in the arid Southwest and has approximately 1 million residents (9. 7% of the residents are > 65 years of age). PM data were obtained from the U.S. Environmental Protection Agency (EPA) National Exposure Research Laboratory Platform in central Phoenix. We obtained gaseous pollutant data, specifically carbon monoxide, nitrogen dioxide, ozone, and sulfur dioxide data, from the EPA Aerometric Information Retrieval System Database. We used Poisson regression analysis to evaluate the associations between air pollution and nonaccidental mortality and cardiovascular mortality. Total mortality was significantly associated with CO and NO(2) (p < 0.05) and weakly associated with SO(2), PM(10), and PM(CF) (p < 0. 10). Cardiovascular mortality was significantly associated with CO, NO(2), SO(2), PM(2.5), PM(10), PM(CF) (p < 0.05), and elemental carbon. Factor analysis revealed that both combustion-related pollutants and secondary aerosols (sulfates) were associated with cardiovascular mortality.     

Measurement of benzene in the workplace and its evolution process, Part I: Overview, history, and past methods

Benzene and its measurement continues to be important in the field of industrial hygiene. This two-part article reviews the history of occupational and environmental sampling and analysis of benzene from the early 1900s to the present. Part I provides an overview and details of the methods used in the past; history of the American Conference of Governmental Industrial Hygienists' threshold limit values for benzene; and reviews portable, grab, and integrated sampling methods as well as the various analytical methods. It is important to review and understand the past in order to predict future trends.