Optimization of microplate-based phenol-sulfuric acid method and application to the multi-sample measurements of cellulose nanofibers.

The phenol-sulfuric acid (PSA) method is a widely used colorimetric method for determining the total saccharides. Microplate-based PSA methods have been developed to handle a large number of samples and reduce the use of hazardous chemicals. However, the optimal procedures and measurement conditions for this method have not yet been fully established. To address this gap, we investigated the optimal procedure for microplate-based PSA. In addition to glucose (Glc), two types of cellulose nanofibers (CNFs) were also evaluated as they are a new type of nanomaterial, and a technique to quantify the concentration of CNFs is required in their safety assessment. The results showed that the thermal reaction with sulfuric acid before the addition of phenol resulted in a higher coloration than was shown after the addition of phenol. Furthermore, the longer the resting time after shaking with phenol, the greater the coloration and smaller the variation, with a resting time of 60 min or longer being optimal. This research provides valuable insights into improving the reliability and efficiency of the PSA method, which can facilitate the analysis of saccharides and other substances in a range of applications.

Potential issues specific to cytotoxicity tests of cellulose nanofibrils.

Cellulose nanofibrils (also called cellulose nanofibers or nanofibrillated cellulose [CNFs]) are novel polymers derived from biomass with excellent physicochemical properties and various potential applications. However, the introduction of such new materials into the market requires thorough safety studies to be conducted. Recently, toxicity testing using cultured cells has attracted attention as a safety assessment that does not rely on experimental animals. This article reviews recent information regarding the cytotoxicity testing of CNFs and highlights the issues relevant to evaluating tests. In the literature, we found that a variety of cell lines and CNF exposure concentrations was evaluated. Furthermore, the results of cytotoxicity results tests differed and were not necessarily consistent. Numerous reports that we examined had not evaluated endotoxin/microbial contamination or the interaction of CNFs with the culture medium used in the tests. The following potential specific issues involved in CNF in vitro testing, were discussed: (1) endotoxin contamination, (2) microbial contamination, (3) adsorption of culture medium components to CNFs, and (4) changes in aggregation/agglomeration and dispersion states of CNFs resulting from culture medium components. In this review, the available measurement methods and solutions for these issues are also discussed. Addressing these points will lead to a better understanding of the cellular effects of CNFs and the development of safer CNFs.

Contaminant microorganisms in the in vitro evaluation of cellular responses of cellulose nanofibers and their microbial inactivation using gamma irradiation.

Cellulose nanofibers (CNFs) are fibrous nanomaterials produced from plants. Since some nanomaterials are toxic, toxicity evaluation, including in vitro examinations using cultured cells, is essential for the effective use of CNFs. On the other hand, microorganisms in the environment can contaminate CNF suspensions. The contamination of CNF samples and the effects of contaminating microorganisms on in vitro examinations were investigated in this study. Microorganism contamination in CNF samples was examined, and microbial inactivation of CNF suspensions using gamma irradiation was evaluated. After gamma-ray irradiation at absorbed doses of 0.5, 1, 5, and 10 kGy, the cellular effects of CNF suspensions were examined using 6 types of cultured cell, HaCaT, A549, Caco-2, MeT-5A, THP-1, and NR8383 cells. CNF samples were contaminated with bacteria and CNF suspensions exhibited endotoxin activity. Gamma irradiation effectively inactivated the microorganisms contained in the CNF suspensions. When the absorbed dose was 10 kGy, the fiber length of CNF was shortened, but the effect on CNF was small at 1.0 kGy or less. CNF suspensions showed lipopolysaccharides (LPS)-like cellular responses and strongly induced interleukin-8, especially in macrophages. Absorbed doses of at least 10 kGy did not affect the LPS-like activity. In this study, it was shown that the CNF suspension may be contaminated with microorganisms. Gamma irradiation was effective for microbial inactivation of suspension for invitor toxicity evaluation of CNF. In vitro evaluation of CNFs requires attention to the effects of contaminants such as LPS.

Air exchange rates and advection-diffusion of CO2 and aerosols in a route bus for evaluation of infection risk.

As COVID-19 continues to spread, infection risk on public transport is concerning. Air exchange rates (ACH) and advection-diffusion of CO2 and particles were determined in a route bus to evaluate the infection risk. ACH increased with bus speed whether windows were open or closed, and ACH were greater when more windows were open. With two open windows, ACH was greater when a front and rear window were open than when two rear windows were open. With both front and rear ventilation fans set to exhaust, ACH was more than double that when both were set to supply. With air conditioning (AC) off, CO2 and particles spread proportionally at the same rate from a source, whereas with the AC on, the spread rate of particles was about half that of CO2 , because particles might be trapped by a prefilter on the AC unit. Infection risk can be reduced by equipping AC unit with an appropriate filter. Calculations with a modified Wells-Riley equation showed that average infection risk was reduced by 92% in the moving bus with windows open comparing to with windows closed. When the bus was moving with windows closed, exhaust fan operation reduced the average risk by 35%.

Quantitative evaluation of carbon nanomaterial releases during electric heating wire cutting and sawing machine cutting of expanded polystyrene-based composites using thermal carbon analysis.

Field measurements were conducted at a facility where expanded polystyrene-based carbon nanomaterial composites, namely, carbon nanotube and carbon black composites, were cut with an electric heating wire cutter or a circular sawing machine. The aerosol particles released during the cutting of the composites were measured using real-time aerosol monitoring, gravimetric analysis, thermal carbon analysis, and scanning electron microscopic observations. This study had two major goals: (1) to quantitatively evaluate the concentrations of airborne carbon nanomaterials during the cutting of their composites; (2) to evaluate the capability of thermal carbon analysis to quantify airborne carbon nanomaterials in the presence of expanded polystyrene-derived particles. The results of thermal carbon analysis showed that the concentrations of elemental carbon (an indicator of carbon nanomaterials) for all the respirable dust samples in both cutting processes were less than the limit of detection (∼2 µg/m3), which is nearly equivalent to or lower than the occupational exposure limits for carbon nanotubes (1 to 50 µg/m3). For total dust, which includes particles larger than respirable size, although the elemental carbon concentrations during heating wire cutting were low (<3 µg/m3), those during sawing machine cutting were up to 58 µg/m3. In scanning electron microscopic observations, micron-sized particles composed of or including carbon nanotubes were detected only in aerosol particles collected during the sawing machine cutting. Therefore, heating wire cutting is considered preferable. This study demonstrated that thermal carbon analysis can quantify airborne carbon nanomaterials in the presence of expanded polystyrene-derived particles.

Risk Assessment of the Carbon Nanotube Group.

This study assessed the health risks via inhalation and derived the occupational exposure limit (OEL) for the carbon nanotube (CNT) group rather than individual CNT material. We devised two methods: the integration of the intratracheal instillation (IT) data with the inhalation (IH) data, and the "biaxial approach." A four-week IH test and IT test were performed in rats exposed to representative materials to obtain the no observed adverse effect level, based on which the OEL was derived. We used the biaxial approach to conduct a relative toxicity assessment of six types of CNTs. An OEL of 0.03 mg/m(3) was selected as the criterion for the CNT group. We proposed that the OEL be limited to 15 years. We adopted adaptive management, in which the values are reviewed whenever new data are obtained. The toxicity level was found to be correlated with the Brunauer-Emmett-Teller (BET)-specific surface area (BET-SSA) of CNT, suggesting the BET-SSA to have potential for use in toxicity estimation. We used the published exposure data and measurement results of dustiness tests to compute the risk in relation to particle size at the workplace and showed that controlling micron-sized respirable particles was of utmost importance. Our genotoxicity studies indicated that CNT did not directly interact with genetic materials. They supported the concept that, even if CNT is genotoxic, it is secondary genotoxicity mediated via a pathway of genotoxic damage resulting from oxidative DNA attack by free radicals generated during CNT-elicited inflammation. Secondary genotoxicity appears to involve a threshold.

Evaluation of particles released from single-wall carbon nanotube/polymer composites with or without thermal aging by an accelerated abrasion test.

To provide data required for assessing the environmental health and safety risks of nanocomposites, abrasion-induced particle release from single-wall carbon nanotube (SWCNT)/polymer composites with or without thermal aging were evaluated by a shot blast system. First, overall composite weight loss (i.e., overall particle release) as a result of shot blasting was measured. Incorporating 5 wt% SWCNTs in polystyrene (PS) matrix was observed to reduce overall particle release by approximately 30% compared with pure PS. Heat treatment of the 5 wt% SWCNT/PS composites at 100°C for 10 days induced very slight change in overall particle release due to shot blasting. However, heat treatment at 350°C for 1 hr greatly deteriorated the abrasion resistance of the composites, enhancing overall particle release. Second, to verify the existence and form of SWCNTs released from the composites, released particles were observed by electron microscopy. Micron-sized particles with protruding SWCNTs and submicron-sized SWCNT clusters were observed in the particles released from the composites. Heat treatment of the composites at 350°C for 1 hr enhanced SWCNT release, which mainly formed clusters or rope-like bundles.

Algal growth inhibition test with TEMPO-oxidized cellulose nanofibers.

Ecotoxicity data on cellulose nanofibers (CNFs) are limited despite their wide potential applications prospects, such as structural and packaging materials, filters, coatings, foods, and cosmetics. In this study, toxicity tests of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized CNFs (TEMPO-CNFs), which are one of the major CNF products commercially available in Japan, on the green alga Raphidocelis subcapitata were conducted. As nanomaterials are considered difficult-to-test substances, the Organisation for Economic Co-operation and Development has released a guidance document that provides considerations regarding ecotoxicity tests of nanomaterials. In the algal growth inhibition tests of TEMPO-CNFs, there were specific issues to be examined, including the effects of medium components on the characteristics of TEMPO-CNFs, CNF interference with algal density measurements, algal interference with CNF measurements, and the effects of ion concentration changes in the test medium by the addition of CNFs on algal growth. To examine these issues, we conducted preliminary studies and established a suitable test method for algal growth inhibition tests of TEMPO-CNFs. We confirmed that the components in the medium for algal growth inhibition tests had negligible effects on the characteristics (zeta-potential, viscosity, and morphology) and concentration stability of TEMPO-CNFs and that in vitro and in vivo fluorescence measurements were applicable for estimating the algal densities, without interference by TEMPO-CNFs. In contrast, we observed that the grown algae interfered with the CNF concentration measurements. Therefore, we established a method to correct the measured CNF concentrations by estimating the algal contribution. Furthermore, we found that the nutrient salt concentrations in the medium changed due to interactions with CNFs; however, this change did not affect algal growth. Based on the results of the preliminary studies, algal growth inhibition tests of TEMPO-CNFs were conducted using in vitro and in vivo fluorescence measurements, along with measurements of CNFs and ion concentrations in the test dispersions. The test results showed that no growth inhibition was observed on growth rate or yield even at the maximum CNF concentration of 100 mg/L, suggesting that the ecological effect of TEMPO-CNFs on algae was relatively low. The results of this study will be valuable for conducting ecotoxicity assessments on additional CNFs and comparable nanomaterials in future studies.

Estimation of microplastic emission and transfer into Tokyo Bay, Japan, using material flow analysis.

To reduce microplastic (MP) discharge into the aquatic environment, it is necessary to properly identify its sources and amounts. Here, specific MP sources, i.e., personal care products (PCPs), fibers from clothes, and tire-wear particles (TWPs) were focused, and MP generations from these sources in the Tokyo Bay watershed, Japan, were estimated based on statistical data on production and reported emission factors of the MP sources and executing considering uncertainty on the data. Potential annual MP emission into Tokyo Bay was estimated to be 10.2 ± 1.6, 38 ± 22, and 1500-1800 tons for PCPs, fibers, and TWPs, respectively. Emissions into Tokyo Bay by assuming MP density and diameter was estimated. For fiber, the fraction to potential emission was estimated at 1.0-2.8 %. This study contributes to determining potential discharge pathways. This will assist in the application of appropriate measures to reduce MP discharge into water bodies.

Quantitative identification of sources of dioxin-like polychlorinated biphenyls in sediments by a factor analysis model and a chemical mass balance model combined with Monte Carlo techniques.

The major sources of dioxin-like polychlorinated biphenyls (PCBs) in two sediment cores from Tokyo Bay and Lake Shinji (both in Japan) were identified and their source contributions estimated using two receptor models. The first was a nonnegative constrained factor analysis (FA) model, and the second was a nonnegative constrained chemical mass balance model combined with Monte Carlo techniques (CMB-MC) to take into account the variability and uncertainty in both PCB congener profiles of sources and environmental samples. According to the FA model, variations in the concentrations of dioxin-like PCBs in each sediment core were accounted for almost entirely by two factors, which were considered to correspond to Kanechlors (KCs; Japanese PCB products) and incineration. The CMB-MC model investigated the trends of the burdens from four types of KCs and incineration to the concentrations of dioxin-like PCBs in each sediment core. The results for both sediment cores obtained by both models indicated that the burden from KCs increased gradually beginning in the 1950s, peaked around 1970, and declined thereafter, whereas the burden from incineration increased gradually from the 1950s to the early 1990s. The estimated contribution from incineration to the toxic equivalent concentration of dioxin-like PCBs was comparable to that from KCs.

Performance evaluation of newly developed portable aerosol sizers used for nanomaterial aerosol measurements.

Nanomaterial particles exhibit a wide range of sizes through the formation of agglomerates/aggregates. To assess nanomaterial exposure in the workplace, accurate measurements of particle concentration and size distribution are needed. In this study, we evaluated the performance of two recently commercialized instruments: a portable scanning mobility particle sizer (SMPS) (NanoScan, TSI Inc.), which measures particle size distribution between 10 and 420 nm and an optical particle sizer (OPS, TSI Inc.), which measures particle size distribution between 300 and 10,000 nm. We compared the data measured by these instruments to conventional instruments (i.e., a widely used laboratory SMPS and an optical particle counter (OPC)) using nano-TiO(2) powder as test aerosol particles. The results showed obvious differences in the size distributions between the new and old SMPSs. A possible reason for the differences is that the cyclone inlet of the new SMPS (NanoScan) acted as a disperser of the weakly agglomerated particles and consequently the concentration increased through the breakup of the agglomerates. On the other hand, the particle concentration and size distributions measured by the OPS were similar to the OPC. When indoor aerosol particles were measured, the size distribution measured by the NanoScan was similar to the laboratory SMPS.

Analysis of atmospheric behavior of PCDDs/PCDFs by a one-compartment box model.

The overall atmospheric behavior of PCDDs/PCDFs in the Kanto region, Japan, was simulated by a one-compartment box model. For each homologue the relative significance and temperature dependences of dry deposition, wet deposition, degradation, and advection in both gas and particulate phases were examined and compared. The results of the model calculation suggested that the rates for dry deposition are comparable to those for wet deposition, and the rates for advection are comparable to those for bulk (dry+wet) depositions in the Kanto region. On the other hand, the rates of degradation for PCDDs/PCDFs in the atmosphere in the Kanto region would be negligible. The emission rates and the bulk deposition fluxes in the entire Kanto region estimated by the model calculation based on observed air concentrations were 0.084-0.90 kg-TEQ/month and 0.045-0.43 kg-TEQ/month, respectively. These estimated emission rates and bulk deposition fluxes were slightly higher than the estimated emission rate based on observed emission concentrations and the estimated bulk deposition fluxes based on observed deposition fluxes collected on water deposition surface, respectively. This study showed the model calculation can be available for understanding of the overall atmospheric behavior, verification of the source inventory, and estimation of deposition flux on the actual environment including various deposition surfaces.

Source and behavior analyses of dioxins based on congener-specific information and their application to Tokyo Bay basin.

Identification of pollution sources and estimation of their contribution to dioxin pollution are important for taking better countermeasures against such sources. The information based on seventeen 2,3,7,8-chlorine-substituted congeners and 10 homologues is usually not sufficient for source identification. The usefulness of detailed congener-specific information of tetra- to octachlorinated polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans was examined by principal component analysis and compared with conventional dioxin information. It was revealed that congener-specific information was far more effective for source identification than conventional information. For source apportioning, it was shown that multiple regression analysis with detailed source congener profiles could be carried out successfully when calculations were performed for individual homologues. This was because of the large variation in homologue composition in source dioxin profiles in addition to the difference in environmental behavior among the homologues. The newly proposed methods for statistical analysis were applied to the estimation of dioxin mass balance in Tokyo Bay basin. It was found that Tokyo Bay sediment core contained dioxins from three major sources: combustion, pentachlorophenol (PCP), and chloronitrophen (CNP). PCP and CNP contained large amounts of dioxin as impurity and were used extensively as paddy field herbicides in Japan in the past. The total dioxin load from the two herbicides to the basin during the past 45 years (1951-1995) was estimated to be five times larger than that from combustion source in terms of toxic equivalents (WHO-TEQ). However, in the surface sediment of the bay, the contribution from the herbicides was nearly equal to that from combustion. The herbicide contribution peaked during the 1960s and 1970s and gradually decreased thereafter.

Quantitative source identification of dioxin-like PCBs in Yokohama, Japan, by temperature dependence of their atmospheric concentrations.

The source and environmental behavior of dioxin-like polychlorinated biphenyls (PCBs) together with other PCBs and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/PCDFs) were evaluated based on analysis of variations in their atmospheric concentrations in Yokohama, Japan. Potential factors responsible for variations in the atmospheric concentrations of the congeners were investigated by principal component analysis and multiple regression analysis of the data. Two major variations were seen: one had strong temperature dependence, while the other had no significant temperature dependence. A possible explanation for this difference is that the former is related to congeners released by volatilization (e.g., volatilization from commercial PCB products and past polluted environments), while the latter is related to congeners emitted from thermal processes. The relative contributions of dioxin-like PCBs released by volatilization and those emitted from thermal processes were estimated based on the temperature dependence of the atmospheric concentrations. The results suggest that both dioxin-like PCBs emitted from thermal processes and those released by volatilization are significant sources of air pollution in this area in terms of the toxic equivalent (TEQ) for dioxin-like PCBs. We demonstrated that the present approach based on variations in atmospheric concentrations can be useful in providing a qualitative as well as quantitative understanding of source information.