Combined application of gamma radiation, cleaning and chemical sanitizers in decontamination of vehicle air conditioning filters

This work aimed to analyze the fungal contamination of air-conditioning filter waste (n=15) as an indicator of Quality Air Indoor from different car models in São Paulo city in São Paulo State, Brazil, during the period from October 2018 to July 2019. Three different treatments were used for the decontamination of car air conditioning filters, such as mechanical vacuum cleaning (I), vacuum cleaning and use of sanitizing product (II), and sanitizing product associated with radiation treatment at a dose of 17 kGy (III). After the treatments, microbiological analyses were performed and samples were plated in Petri dishes containing Sabouraud agar transferred by Swabs, and incubated for 7 days at 25 °C. The Petri dishes were stored in a standard Biochemical Oxygen Demand incubator, for the growth of fungal cultures. After incubation, the fungal cultures were evaluated, and the fungal counting was expressed in unit-forming colonies (UFC) and frequency in samples (%). The fungi were examined by lactophenol blue solution staining for microscopy. All samples of treatment I and II were contaminated with various fungal genera and high bioburden, namely (treatment I) Alternaria alternata, Aspergillus flavus, A. niger, Cladosporium spp., Fusarium spp., Mucor spp., Nigrospora spp., Not Sporulated Fungi (NSF), Penicillium spp., Rhizopus spp., Rhodotorula spp., Trichoderma spp. and yeasts. Treatment II showed Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, A. ochraceus, Cladosporium spp., Mucor spp., NSF, Penicillium spp., Phoma spp., Rhizopus spp., Rhodotorula spp., Trichoderma spp., and yeasts. Treatment III presented NSF and yeasts, with 80% of material decontamination.


INTRODUCTION
Indoor air quality is an indicator of environmental health that takes into account thermal comfort, factors that interfere in precarious air conditions, such as the presence of fungi, bacteria and carbon dioxide in indoor air-conditioned environments [1].
In a study performed by Zulauf et al. [2] that determined particulate matter (PM0.3 and PM2.5), fungi, and airborne bacteria concentrations. This search was carried out during a routine travel trip of 51 cars, in eastern Germany and rural areas. The mean bacterial count was 350 colony-forming units (CFU) m −3 and mean fungal count was 13 CFUm −3 . Barnes et al. [3] found a significant positive correlation (0.30, < 0.05) between bacterial counts and particulate matter (PM 2.5).
The lack of studies on air quality within automotive passenger vehicles in the downtown area of São Paulo, one of the most polluted cities in the world and with the largest fleet of vehicles in Brazil, was the objective of the study of Aquino et al. [1]. They aimed to analyze the fungal contamination in air-conditioning filters collected from twenty-one automotive vehicles and the results of the study showed seventeen fungal genera in all samples collected (100%), including toxigenic fungi such as Penicillium, Fusarium and Aspergillus. Such an indoor air quality may compromise the health of a portion of the population, such as professional drivers.
According to IAEA [4], the combinations of the processes and their applications are being pursued to meet the end objectives of improved decontamination, waste volume reduction, safety and overall cost-effectiveness in the irradiation treatment. According to Thakur and Singh [5], the use of combined processes has been found to inhibit the development of undesirable changes caused by irradiation into substrate material. Almost all standard production car filters are paper, but filter elements are also generally made from different materials: paper, cotton, or foam. Inlet air filtration systems in modern passenger car engines use single-stage filters with panel filters made of pleated filter paper, which are porous materials, characterized by particle separation efficiency (≥ 5 µm), filtration performance of 99.9%, and low thickness (0.4-0.8 mm). Filter paper retains dust particles at the fibers of a porous filter media as a result of different forces and separation mechanisms [6].
American Society of Mechanical Engineers (ASME), Nuclear Regulatory Commission (NRC) and American Society for Testing and Materials (ASTM) published documents with specifications that combine to basic physical, chemical, test, and performance standards for filter media and impregnated activated carbon as well as for assembled componentes. Aging mechanisms and effects are discussed in conjunction with stressors, the agents, or stimuli that can result degradation [7,8].
DeAngelis and cols. [9] observed that filtration performance was degraded after 25 kGy in different models of N95 respirators (mask filter). Electrostatic potential was measured on each respirator layer and statistically significant changes were observed in layers with electret filter media.
One way to prevent these changes in constituents of different irradiated materials is to reduce the radiation doses and use combined irradiation with heating, chemicals, cryogenic temperature, and a modified atmosphere. The decreased cost of irradiation at lowered absorbed doses may offset the additional cost of any other applied process, depending upon the cost of the process relative to the cost of irradiation. In this study it is convenient to try previous cleaning and chemical treatment of 15 filters of vehicles before irradiation in order to use 17 kGy of dose of radiation. Soriani et al.
[10] after using gamma radiation at the dose of 17.8 kGy showed that the content of the main chemical active principles of raw plant materials was not modified and controlled high microbial bioburden. Denatonium benzoate (C28H34N2O3) is a substance of chemical classes of nitrogen compounds that is used in cleaning car filters. denatonium benzoate appears to be safe when used at low concentrations and has been used as an alcohol denaturant [11,12].

Sampling and fungi isolation
Fungal isolation was entirely based on inoculation in Petri dishes with swabs directly in Sabouraud agar and the incubation of samples during 7 days at 25 ºC was under carefully standardized conditions, according to Pitt and Hocking methods [13] in a Biochemical Oxygen Demand (BOD) incubator.
The fungal identification was performed using lactophenol cotton blue solution for microscopy, for staining molds, as described by Pitt and Hocking [13]. After the fungal analysis of control group, 15 filter samples were cleaning with a vacuum system and treated by sanitizer for vehicle air conditioning filters. The air conditioner sanitizer spray was used in the samples for 5 minutes, and they were composed by denatonium benzoate and were kept in bags ( Figure 1) with an atmosphere filled with sanitizer spray and kept in cardboard boxes before the gamma radiation treatment. After all treatment procedures, the samples were analyzed in Petri dishes, in triplicate ( Figure  2), to fungal counting, expressed as colony-forming units (CFU) and percentage (%) [13].

Irradiaton at cobalt source
The filters samples were individually protected in plastic bags (   Two samples showed 2 CFU (colony-forming units) of NSF and one showed 1 CFU of yeasts in treatment III. Table 1 shows the decrease of fungal burden data (%) of treatment III in comparison with treatment I and II.
The fungal growth was favored by high humidity conditions in samples that were sprayed with sanitizer, that did not inhibit the fungal contamination. According to Reponen and cols. [17], the relative humidity changes the spore size. The same authors reported that the highest change of the aerodynamic diameter was found in Cladosporium cladosporioides spores, that increased from 1.8 μm to 2.3 μm when the relative humidity increased from 30% to ∼ 100%. The size increase corresponds to an approximate doubling of the particle volume [17]. Udaya et al. [15] isolated 17 genera from the surface of bus seats. Aspergillus spp. was most frequently represented inside the vehicle. Viegas et al. [16] reported that Cladosporium spp. was the most prevalent fungal species from taxi filters in three cities of Portugal (Lisbon, Loures and Setúbal). In this present study in São Paulo city, the Aspergillus genera was predominant, after NSF and Cladosporium spp. On the other hand, treatment III, which used the denatonium benzoate (spray) in association with gamma radiation (17 kGy) showed complete decontamination in 80% of samples. No fungal growth was observed in 12 filter samples (1, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14, and 15). Only 3 samples (filters 2, 6, and 11) presented the lowest initial bioburden, as demonstrated in Figure 6, due to the fact that the filters were cleaner at the time of replacement. The effect of water radiolysis plus the chemical effect of denatonium benzoate justified the reduction of bioburden in Treatment III because of higher humidity, with the use of the liquid spray in samples. The indirect effect of the ionizing radiation is associated with water presence in the substrate. The direct effect causing direct damage to biomolecules, such as double-strand breakage in DNA, and provoking genotoxic DNA alterations and cell damage [18]. The liquid spray of denatonium benzoate is associated with water radiolysis (indirect effect of radiation) and leads to the production of free radicals (e−aq, ·OH, H·, and HO2·) and molecular products (H2, H2O2), that are referred to as primary products of radiolysis [19].
They are usually classified into reducing (e−aq, H·) and oxidizing (·OH, HO2·, H2O2) equivalents. With a longer lifetime, hydroxyl radical (·OH) is the most effective oxidant radical towards biological molecules: it has a high standard potential (E° = 2.34 V) with respect to Standard Hydrogen Electrode (SHE) at neutral pH [19]. The primary products of radiolysis are capable of damaging fungal cells causing growth inhibition.

CONCLUSION
The methods of vacuum cleaning and sanitizer spray with denatonium benzoate were not efficient to fungal control. The association with radiation, 17 kGy, showed that 80% of samples were completely decontaminated.
Two samples demonstrated only the growth of yeasts (not pathogenic fungi). No mycotoxigenic fungi such as Aspergillus, Penicillium, and Fusarium genera were detected in irradiated samples using combined methods.
In order to establish a protocol with two combined methods for the control of fungi in air filters, the use of gamma radiation and sanitizer products showed that it is an efficient way to control mycotoxigenic or pathogenic fungi, using low doses to recycle the material against radiorresistant species.