Sugita et al. Genes and Environment (2019) 41:7 https://doi.org/10.1186/s41021-019-0120-0 RESEARCH Open Access Evaluation of the genotoxicity of PM2.5 collected by a high-volume air sampler with impactor Kazutoshi Sugita1*, Yuka Kin1, Mayuko Yagishita2, Fumikazu Ikemori3, Kimiyo Kumagai4, Toshihiko Ohara5, Makoto Kinoshita6, Kazuyuki Nishimura2, Yukihiko Takagi1 and Daisuke Nakajima7 Abstract Background: The harmful effects of fine particles with an aerodynamic diameter less than 2.5 μm (PM2.5) on respiratory organs are emphasized in pollution studies because PM2.5 have high deposition rates in the respiratory organs and contain various hazardous compounds. In this study, a sampling method combining a high-volume air sampler (HV) with a PM2.5 impactor was developed for collecting large quantities of PM2.5. The concentrations of elemental carbon (EC), organic carbon (OC), inorganic ions, and polycyclic aromatic hydrocarbons (PAHs) were measured in PM2.5 collected by the high-and low-volume air samplers (LV). Results: Similar results were obtained from the HV and LV methods, with respect to inorganic carbon, organic carbon, sodium ions, ammonium ions, and PAHs with more than four rings. Because of the much larger amount of PM2.5 could be collected by the HV method, the trace constituents, that were difficult to detect by the conventional LV method, were readily detected by the HV method. Furthermore, when the microsuspension method that was modified more sensitive Ames mutagenicity test, was used to test the PM2.5 samples at four sites, mutagenic activities were detected by strains TA100 and TA98. Most of the mutagenic activity was associated with the PM2.5 fraction and mutagenic activity in winter was greater than that in summer. Conclusions: The HV method produced results similar to those from the conventional LV method with respect to the PM2.5 components present in the atmosphere in relatively high concentrations, but its 40-fold greater flow rate enabled the detection of mutagenic compounds present in only trace concentrations. Keywords: PM2.5, High-volume air sampler, Impactor, Mutagenic activity, Microsuspension, TA98, TA100, Dibenzo [a,l]pyrene Background al. 2016). PM2.5 are widely studied because of their harm- Recent reports in Japan have indicated decreases in the ful influence on human health, such as cardiovascular dis- emission of suspended particulate matter (SPM) [40], ease and respiratory disorders, as a result of the high polycyclic aromatic hydrocarbons (PAHs) [7] and dioxins deposition rate in the respiratory organs [28]. Various epi- into the atmosphere, by emission control on car engines demiological and toxicological studies have shown strong and improved management of waste incineration. On the positive correlations between PM2.5 concentrations in the other hand, it has been reported that the Japanese atmos- atmosphere and the death rate by respiratory system dis- phere is influenced by sulfur oxides from coal combustion ease [8], and a direct influence on collective human mor- from China [30], so there is considerable interest in Japan tality [4, 26, 27]. Furthermore, PAHs, such as benzo [a] regarding air pollution by PM2.5 from China ([39], Ling et pyrene (BaP, [1, 3, 22, 29]), nitro-PAHs, and dioxins [28], which are related compounds with carcinogenic activity, have been detected in PM2.5. PAHs derived from atmos- * Correspondence: [email protected] pheric suspended particulates were shown to be retained 1Azabu University, 1-17-71, Fuchinobe, Chuou-ku, Sagamihara, Kanagawa 252-5201, Japan in the lungs of a dog [32]. The World Health Organization Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sugita et al. Genes and Environment (2019) 41:7 Page 2 of 11 (WHO) has set guidelines for PM2.5 in the atmosphere at phosphate potassium, sodium hydroxide, D-glucose (The 10 μg/m3 for the annual average concentration and at reagent best quality, FUJIFILM Wako Pure Chemical 25 μg/m3 for the daily average. The continuous monitor- Corporation, Oosaka, Japan) and agar (Nacalai Tesque Inc., ing of PM2.5 in Japan is essential, and an environmental Kyoto, Japan) were used for a mutation assay. standard of PM2.5 in the atmosphere was set in 2009 at S. typhimurium strains TA98 and TA100 were distrib- 15 μg/m3 for the annual average and at 35 μg/m3 for the uted from National Institute of Public Health. daily average, and continuous monitoring is carried out in all parts of Japan. PM2.5 is collected by LV as part of the PM2.5 sampling monitoring, but chemical analysis of the samples collected The PM2.5 samples were collected at four locations in is limited because only small samples can be collected by Japan, namely Maebashi (East longitude 36.4045°, North this method, which may be below the detection limit of latitude 139.0961°), Tsukuba (East longitude 36.0498°, some pollutants. Therefore, equipment collecting larger North latitude 140.1175°), Nagoya (East longitude 35.0990°, amounts of PM2.5 is needed. North latitude 136.9156°) and Fukuoka (East longitude It is currently impossible to measure all the potentially 33.5948°, North latitude 130.3646°). At Nagoya, parallel carcinogenic substances contained in PM2.5 samples be- sampling by the LV and HV methods was carried out to cause of the large number of compounds present at very measure the concentrations of EC, C and some ion compo- low concentrations. Therefore, to achieve a comprehen- nents in the spring and summer 2012 and winter 2013 sive evaluation of the harmful effects of substances associ- (21 samples) and to measure the concentrations of PAHs in ated with PM2.5, a mutagenic assay, such as the Ames the summer 2013 and winter 2014 (15 samples). At the test, is needed. On the other hand, the conventional Ames other sampling points, sampling by the HV method was mutagenicity test uses Salmonella typhimurium strains carried out to measure the mutagenic activities in the sum- TA98 (in which frameshift mutations are detected) and mer 2012 and winter 2013 (64 samples including 14 sam- TA100 (in which base substitution mutations are detected) ples in Nagoya). which cannot detect mutagenic activity because the sensi- Samples of PM2.5 were collected on 47 mmφ quartz-fiber tivity is not high enough, and the amount of PM2.5 col- filters (PALLFLEX 2500QAT-UP, PALL corporation, New lected is limited. Therefore, modified Ames tests have been York, USA) for a 24 h period by the LV method and on developed in which the mutagenicity of PM2.5 samples has 20.3 × 25.4 cm quartz-fiber filters (PALLFLEX 2500QAT-UP, been increased using TA98 and a TA98-derivative strain, PALL corporation, New York, USA) by the HV method. A YG1024, with elevated O-acetyltransferase levels. The low-volume air sampler, Partisol 2000-FRM Air Sampler YG1024 strain shows hyper-responsiveness to nitro-PAHs (Thermo Fisher Scientific, Massachusetts, USA) was used and amino-PAHs [6, 36], but there is the risk of introdu- with a flow rate of 16.7 L/min as LV method. In 2000-FRM, cing a bias toward these compounds when this modified PM2.5 were collected by back-up filter, and PM > 2.5 were Ames test is used. eliminated by impactor that consist of filter and pump oil In the current study, parallel sampling of PM2.5 was [25]. This PM 2.5 collection method is certified by the carried out using the HV and LV methods, with concen- Federal Reference Law of the United States and is consistent trations of EC, OC, some ion components, and PAHs be- with the manual of the Japanese Ministry of the Environ- ing compared in PM2.5 collected by the two sampling ment. A high-volume air sampler, HV-700R (SIBATA methods, to evaluate the validity of collecting PM2.5 using Scientific Technology Ltd., Saitama, Japan) was used with a the new HV method. Subsequently, PM2.5 and the par- flow rate of 700 L/min as HV method. In the HV method, ticle bigger than PM2.5 (PM > 2.5) were collected using an impactor for PM2.5 (Custom-made based on a HV-100 the HV method, and the carcinogenic potential was evalu- 2.5 impactor, SIBATA Scientific Technology Ltd., Saitama, ated using the TA98 and TA100 strains, these being the Japan) was mounted on HV and PM2.5 was collected on strains used for the conventional Ames test, to achieve a pre-treated back-up quartz-fiber filters, and PM > 2.5 was comprehensive evaluation of the carcinogenesis-related collected on slit quartz-fiber filters (2500QAT-UP, PALL substances in PM2.5 and PM > 2.5 collected by the HV corporation, New York, USA) referring Fig. 1. Each method. quartz-fiber filter was heat-treated at 450 °C for 2 h in a muffle furnace to remove organic pollutants and then Methods equilibrated in a desiccator at a constant 50% humidity Materials and weighed. Acetone (HPLC grade; FUJIFILM Wako Pure Chemical Corporation, Oosaka, Japan) and dimethyl sulfoxide Analysis of carbonaceous and ion components (DMSO, Dojindo Molecular Technologies, Inc., Kumamoto, A 1.0 cm2 sample of the quartz-fiber filter with aerosol Japan) were used for extraction.