Inhalation Exposure

Carcinogenic and mutagenic risk assessments^{15 (link),60 (link)–63 (link),67 (link)–69 (link)} induced by inhalation of PM2.5-bound enriched with selected nitro-PAHs (1-NPYR, 2-NPYR, 2-NFLT, 3-NFLT, 2-NBA, and 3-NBA) and PAHs (PYR, FLT, BaP, and BaA) were estimated in the bus station and coastal site samples according to calculations done by Wang et al.^{60 (link)}, Nascimento et al.^{61 (link)}, and Schneider et al.^{67 (link)} PAH and PAH derivatives risk assessment is done in terms of BaP toxicity, which is well established^{67 (link)–73 (link)}. The daily inhalation levels (E_I) were calculated as: $$E_I=Ba{P}_{eq}\times IR=(\sum C_i\times TE{F}_i)\times IR$$ where E_I (ng person⁻¹ day⁻¹) is the daily inhalation exposure, IR (m³ d⁻¹) is the inhalation rate (m³ d⁻¹), BaP_eq is the equivalent of benzo[a]pyrene (BaP_eq = Σ C_i × TEF_i) (in ng m⁻³), C_i is the PM2.5 concentration level for a target compound i, and TEF_i is the toxic equivalent factor of the compound i. TEF values were considered those from Tomaz et al.^{15 (link)}, Nisbet and LaGoy^{69 (link)}, OEHHA⁷² , Durant et al.^{73 (link)}, and references therein. E_I in terms of mutagenicity was calculated using equation (1), just replacing the TEF data by the mutagenic potency factors (MEFs) data, published by Durant et al.^{73 (link)}. Individual TEFs and MEFs values and other data used in this study are described in SI, Table S4.
The incremental lifetime cancer risk (ILCR) was used to assess the inhalation risk for the population in the Greater Salvador, where the bus station and the coastal site are located. ILCR is calculated as: $$ILCR=(E_I\times SF\times E_D\times cf\times EF)/(AT\times BW)$$ where SF is the cancer slope factor of BaP, which was 3.14 (mg kg⁻¹ d⁻¹)⁻¹ for inhalation exposure^{60 (link)}, EF (day year⁻¹) represents the exposure frequency (365 days year⁻¹), E_D (year) represents exposure duration to air particles (year), cf is a conversion factor (1 × 10⁻⁶), AT (days) means the lifespan of carcinogens in 70 years (70 × 365 = 25,550 days)^{70 ,72} , and BW (kg) is the body weight of a subject in a target population⁷¹ .
The risk assessment was performed considering four different target groups in the population: adults (>21 years), adolescents (11–16 years), children (1–11 years), and infants (<1 year). The IR for adults, adolescents, children, and infants were 16.4, 21.9, 13.3, 6.8 m³ day⁻¹, respectively. The BW was considered 80 kg for adults, 56.8 kg for adolescents, 26.5 kg for children and 6.8 kg for infants⁷⁰ .

An inhalation exposure system, containing a fluidized-bed powder generator, an animal chamber, and several aerosol monitoring devices, was developed for continuous generation and monitoring of ultrafine or fine TiO₂ aerosols for rodent exposure [15 ]. A schematic of the system is presented in Figure 1. The system was designed based on the criteria of simplicity, ability to disperse fine/ultrafine TiO₂ aerosols, and ease of maintenance. The ultrafine and fine TiO₂ powders were obtained from DeGussa (Aeroxide TiO₂, P25, primary particle size 21 nm, Parsippany, NJ) and Sigma-Aldrich (titanium (IV) oxide, 224227, primary particle size 1 μm, St. Louis, MO), respectively. To reduce the potential formation of agglomerates due to van der Waals force, the TiO₂ powders were carefully prepared for generation by sieving (to remove the large agglomerates), drying (to avoid agglomerate formation due to high humidity), and storage (to prevent agglomerate attraction through contact charges). A fluidized-bed aerosol generator was used in this study because it was able to disperse powders effectively. A 19-liter metabolism chamber that contains an animal cage was modified for use as the whole-body exposure chamber. The cage can accommodate 3 rats for each exposure. During exposure, TiO₂mass concentrations were continuously monitored with a Data RAM (DR-40000 Thermo Electron Co, Franklin, MA) and gravimetrically measured with Teflon filters. Aerosol concentrations between 1.5 and 20 mg/m³ were achieved by adjusting the powder feed rate in the generator. Pulmonary deposition was estimated by the formula: Pulmonary Load = aerosol concentration × minute ventilation × exposure duration × deposition fraction, where minute ventilation and deposition fraction were estimated to be 200 cc and 10%, respectively. The deposition fraction of 10% was based upon Kreyling's alveolar deposition curve for inhaled ultrafine particles in the rat [16 ]. The particle size distributions of TiO₂ aerosols were measured using a cascade impactor (MOUDI, MSP Co., Shoreview, MN), an electrical mobility classifier (SMPS, TSI Inc., Shoreview, MN), and an aerodynamic sizing instrument (APS, TSI Inc.). The impactor was used for measuring mass-based aerodynamic size distributions, while the latter two sizing devices were combined for determining number-based mobility size distributions. In addition, temperature, relative humidity, and pressure in the chamber were monitored throughout the exposure.
To verify that containment in the exposure chamber did not cause an unintended biologic response, Sham/Control exposures (that matched the exposure duration of the experimental groups) were performed throughout the course of these experiments. Because no discernable systemic or microvascular effect was observed in any condition, the data from these experiments were combined into a single "Sham/Control" group. Furthermore, the systemic and microvascular responses of this "Sham/Control" group were not different from those observed in naive rats (data not shown).

Diesel exhaust particles (DEP; NIST 1650b, pH 6.6–6.8) or particulate matter (PM; NIST 1649b) dissolved in sterile PBS (Cat# D8537, Sigma), or PBS alone as control, were administered either to the lung by intratracheal instillation or to the gut by gavage starting at 5–6 weeks of age until sacrifice (for a detailed characterization of the chemical composition see embedded link). Intratracheal instillation was performed as previously described [57 (link)]. Suspension characteristics of the dissolved particles see Additional file 1: Table S2.
For gavage, mice received daily 12 µg DEP or PM (or 60 µg; dose escalation experiment, see Additional file 1: Figure S1) suspended in 200 µL sterile PBS or PBS as control. In the “prestressed” condition, 20 µg DEP daily were used. The rationale for the slightly higher dose in the “prestressed” setting was that we hypothesized that the time frame to develop glucose intolerance would be shorter in a “prestressed” setting induced by HFD/STZ. We therefore assumed a shorter exposure period and chose a slightly higher weekly exposure dose to approximate the total deposition dose of the standard diet model. While gavage was performed 5 days a week, intratracheal instillation was conducted only twice weekly. To keep both models comparable, the same weekly dose of total 60 µg or 100 µg (in case of diabetic mice) DEP or PM, respectively were instilled. The lower dose represents an average daily dose of 8.6 µg/mouse and approximately equates a daily inhalation exposure of about 160 µg/m³ (calculated by the daily exposure divided by the daily inhaled air volume).
The calculation of the daily inhaled air volume was based on a minute volume-to-body weight ratio of 1.491 (L/(min*kg)): $$1.491\frac{L}{min\ast kg}\ast 0.025kg\ast 60min\ast 24h=53\frac{L}{\mathit{day}}=0.053\frac{m^3}{\mathit{day}}$$

Different pollution evaluation indices, including the enrichment factor (EF), geo-accumulation index (I_geo), contamination factor (CF_i), degree of contamination (C_d), pollution load index (PLI), and, potential ecological risk evaluation index (PERI) were used in this study to assess the degree of pollution, details presented in Table 2 and pollution/risk evaluation category are given in Table S2.Table 1

Pollution evaluation indices.

Table 1

Evaluation indices	Equation	Explanation	Reference
Enrichment factor (EF)	EF=(CxMFe)sample(CxMFe)background (1)	(Cx/ MFe) sample = the ratio of the concentration of toxic metals (Cx) to that of iron (MFe) in the soil sample(CM/CFe) background = the same reference ratio in the pre-industrial sample	Chakraborty et al. [42] (link), Islam et al. [7] (link), Sutherland [43]
Geo-accumulation index (Igeo)	Igeo=Log2Cxi1.5×Bxi (2)	Cxi = the studied toxic metal concentration of the ith parameterBxi = the geochemical background value of the toxic metal of the ith parameter in the preindustrial soil samples of the study area	Muller [44] , Islam et al. [45] (link)
Contamination factor (CFi)	CFi=CxiBxi (3)		Hakanson [46] (link)
Degree of contamination (Cd)	Cd=∑i=1nCFi (4)	Cd = the degree of contamination	Hakanson [46] (link)
The pollution load index (PLI)	PLI=CFi1×CFi2×CFi3…………×CFinn(5)	n = number of studied metals	Tomlinson et al. [47] (link)
Potential ecological risk evaluation index (PERI)	Eri=Tri×CFi,PERI=∑i=1nEri (6)	Eri = the particular metal-induced ecological risk index of the ith parameterTri = the biological toxic factor of the ith parameterThe study used the Tri value of As, Ni, Pb, Cd, Cr, and Cu as 10, 6, 5, 30, 2, and 5, respectively	Hakanson [46] (link)

Table 2

Human health risks assessment methods via ingestion, dermal contact and inhalation exposure routes.

Table 2

Assessment approaches	Equation
Chronic daily intake (CDI) (mg/kg/day)	CDIingest=CS×IRS×EF×EDBW×AT×CF(7)CDIdermal=CS×SA×AF×ABS×EF×EDBW×AT×CF(8)CDIinhale=CS×InhR×EF×EDPEF×BW×AT (9)
Hazard quotient (HQ)	HQingest=CDIingestRfD (10)HQdermal=CDIdermalRfD(11)HQinhale=CDIinhaleRfD(12)
Hazard index (HI)	HI=∑HQ=HQingest+HQdermal+HQinhale(13)
Carcinogenic risk (CR)	CRingest=CDIingest×CSFi (14)CRdermal=CDIdermal×CSFi (15)CRinhale=CDIinhale×CSFi (16)Lifetime cancer risk=CRingest+CRdermal+CRinhale (17)

This study estimates the human health risks (non-carcinogenic and carcinogenic) due to exposure to contaminated soil via ingestion, dermal contact, and inhalation exposure pathways for children and adults. USEPA [48] , [39] proposed health risk assessment method was applied (Table 2), and model parameters and references dose are presented in Table 3 and Table S3, respectively.Table 3

Exposure parameters and their values were adopted from USEPA [49] , [50] .

Table 3

Parameter	Unit	Child	Adults
Body weight (BW)	kg	15	70
Exposure frequency (EF)	days/year	350	350
Exposure duration (ED)	years	6	30
Ingestion rate (IR)	mg/day	200	100
Inhalation rate (IRair)	m3/day	10	20
Skin surface area (SA)	cm2	2100	5800
Soil adherence factor (AF)	mg/cm2	0.2	0.07
Dermal Absorption factor (ABS)	none	0.1	0.1
Dermal exposure ratio (FE)	none	0.61	0.61
Particulate emission factor (PEF)	m3/kg	1.3 × 109	1.3 × 109
Conversion factor (CF)	kg/mg	10−6	10−6
Average time (AT)
For carcinogens	days	365 × 70	365 × 70
For non-carcinogens		365 × ED	365 × ED