Urinary 1-hydroxypyrene and Skin Contamination in Firefighters Deployed to the Fort McMurray Fire

Abstract Background In May 2016, firefighters from the province of Alberta, Canada deployed to a fire that engulfed the urban area of Fort McMurray. During the first days of the fire, firefighters experienced heavy smoke exposures during greatly extended work shifts. Urinary samples were collected post-deployment from three fire services for estimation of 1-hydroxypyrene (1-HP) concentration, reflecting exposure to polycyclic aromatic hydrocarbons (PAHs), to determine the effects of respiratory protective equipment (RPE) and skin hygiene in reducing internal dose Methods Urine samples from one fire service (n = 62) were analyzed for 1-HP by two laboratories, using different assays (LC-MS/MS: GC-MS): remaining samples were analyzed just by LC-MS/MS. A Skin Exposure Mitigation Index (SEMI) was computed from questions on opportunities for changing clothing, showering, and washing during breaks. Regression analyses, using 1-HP ng/g creatinine as the dependent variable, assessed the effect of RPE and skin factors on PAH absorption, allowing for environmental exposure and potential confounders. Stratification identified key groups with equal delay in sample collection. Results 1-HP was detected in 71.0% of 62 samples by LC-MS/MS and 98.4% by GC-MS, with good mutual agreement between the methods. In 171 post-fire samples, 1-HP corrected for creatinine was related to current cigarette smoking and recent barbeque. Among those with samples collected within 48 h, urinary 1-HP was correlated with estimated exposure(r = 0.53, P < 0.001). In those with only one rotation before urine sample collection, no effect was seen of RPE use but I-HP was significantly lower (P = 0.003) in those with those with a high score on the SEMI scale, indicating better access to factors mitigating skin absorption. Conclusion Skin exposure to PAHs is an important route of absorption in firefighters, which can be mitigated by good skin hygiene.


LC-MS/MS
LC-MS/MS analysis was performed using an Agilent 1200 high performance liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with a Sciex 5500 Q-trap mass spectrometer (AB Sciex, Concord, Ontario, Canada). Separation of 1-hydroxypyrene was achieved on an Agilent SB-C18 column (Poreshell 120, 3.0X100 mm, 2.7μm) using isocratic elution of water (40%) and acetonitrile (60%) at a flow rate of 0.4 ml/min. The injection volume was 10 µL and the column was kept at room temperature. The mass spectrometer was operated in negative MRM mode. The source temperature was 550 °C with ion spray potential of -4500v, curtain gas at 20, gas 1 at 45, gas 2 at 55 and CAD at high. The MRM transition for the detection of 1-OH-pyrene was 217.0/188.9 with collision energy (CE) at -45 and declustering potential (DP) at -140. The MRM transitions for 1-OH-Pyrene-d9 (ISTD) was 226.1/198.1 with CE at -48 and DP at -150. Data processing was conducted using Multiquant3.0.1 (AB Sciex, Canada).
Sample preparation involves an enzyme deconjugation, followed by protein precipitation and dilution. To an aliquot of 1 mL of urine, 5 µL of ascorbic acid (1.5M), 10 µL of internal standard (1-OH-pyrene-d9, 100 ng/mL), 200 µL of pH5.2 buffer and 10 µL of diluted β-glucuronidase/sulfatase were added. The mixture was incubated on a shaking water bath for 2 hours at 60 °C and then cooled to room temperature. 100 µL of hydrolyzed sample was aliquoted and further diluted with 5uL of ascorbic acid and 900 µL of elution solvent (ACN/pH6 buffer, 0.1M, 2:1)). The mixture was vortexed and then centrifuged at 4000 rpm for 10 min. The supernatant was transferred to a 2 mL silanized autosampler vial for LC-MS/MS analysis.

Quality control and Data Analysis
A set of matrix matched calibrators were prepared by spiking known amounts of 1-OH-pyrene into Surine® negative urine from 0.02 ng/mL to 10 ng/mL. Two in-house QCs were prepared at 0.1 ng/mL and 0.5 ng/mL by a different analyst. A standard reference material® (SRM 3673) which contains 0.029 ng/mL of 1-OHpyrene in urine was purchased from National Institute of Standards & Technology (NIST) and used as an outsource QC. Each batch consisted of 40 Fort McMurray samples, a set of calibrators, a solvent blank, a urine blank, two in-house QCs and SRM 3673. All blanks, calibrators and QCs were hydrolyzed and cleaned up along with the samples. The Blanks and QCs were run after calibrators, in the middle of the sequence and at the end of sequence to insure all samples were bracketed with blanks and QCs. QCs must be within ±20% of targeted value. The quantification of 1-OH-pyrene in the samples was calculated based on the peak area ratio of 1-OH-pyrene to internal standard in the sample against the calibration curve constructed from the calibrators. The retention time of 1-OH-pyrene in the sample must be within ± 0.1min of that in the calibrators. The batch was accepted when all QCs passed and all the blanks were clean.

Method validation
Surine® artificial negative urine was used to validate the method because blank urine from non-smokers may still contain trace levels of 1-OH-pyrene. Method validation was conducted by spiking Surine® negative urine with known amounts of 1-OH-pyrene and 1-OH-pyrene-d9 as internal standard, then hydrolyzed and cleaned up following the procedure described above. It was observed that 1-OH-pyrene was linear up to 500 ng/mL with a correlation coefficient (R 2 ) greater than 0.995. Fig.1 shows a representative calibration curve from 0.02ng/mL to 10 ng/mL. The limit of detection (LOD) and limit of quantification (LOQ) were defined as the concentration of 1-OH-pyrene at which the signal-to-noise ratio (S/N) is equal to or greater than 3 and 6. The method LOD and LOQ are 0.01 ng/mL and 0.02ng/mL respectively. Fig 2 depicts representative chromatograms of the internal standard and 1-OH-pyrene in a human urine sample.
It is well known that LC-MS/MS is prone to ion suppression or enhancement due to the presence of matrix components. Matrix effect (ME) was evaluated in blank urine from different volunteers. Ion suppression was less than 10%.
Recovery, accuracy and precision of spiked urine samples was performed at concentrations of 0.1 ng/mL, 1 ng/mL and 10 ng/mL. The results were summarized in the NIST SRM® 3673 is a urine based certified reference material which contains 0.0293ng/mL of 1-OH-Pyrene. It was used to evaluate the accuracy at near LOQ. The accuracy was 88.1% with coefficient of variation (CV) of 5.1%.

Supplementary materials 2 A) Estimation of environmental smoke concentration (total PM2.5) encountered by each firefighter during the key rotation.
Potential exposures were calculated for the 'key' deployment. This was the first deployment for all but 4 firefighters from fire station A, who gave information only for their most recent deployment. Elements used for this calculation were as follows

1) Data available from Alberta Environment and Parks
Daily 24 Estimates were also obtained for 3 further locations (the airport, the village of Anzac and the area of Mildred Lake) as these were reported by firefighters as additional areas in which they spent time during their key deployment. All estimates except those for Mildred Lake used data from Alberta Environment monitoring stations. The estimates for Mildred Lake use Blue Sky estimates (a combination of air samples taken at different locations and satellite imagery).
2) Total hours on active duty reported by the fire fighter during the key rotation.
The number of hours worked on each day of each shift during the key deployment was calculated.
For example: for firefighter 000 shift 1 started at 9h00 on May 3 rd with reported length 23 hours; he will have worked from 9h00 to 23h59 on May 3 rd so 15 hours for May 3 rd and he would have worked 9 hours on May 4 th . This was repeated for every shift as to determine how many hours were worked on what days for their entire key deployment.
3) The percentage of time spent in each location during the key deployment (as reported by the firefighter) This was used to compute a time weighted exposure for each firefighter for each day of deployment.
4) The total time was used to adjust the time weighted estimate PM2.5 estimates were adjusted to reflect 100% of their day because many firefighters either overestimated or underestimated the amount of time they spent in each location (i.e the total percentage exceeded or fell short of 100%) Example: (535*5*.1) + (320*5*.8 )=1547.5 for 90% of key deployment adjusted as 547.5/0.9=1719.44 5) A cumulative exposure for their key deployment was obtained by summing over all shifts B) Calculation of total exposure to PM 2.5 across multiple deployments 1) For all firefighters that had multiple deployments to Fort McMurray between May 1 st and June 30 th 2016, no specific information as to where they worked and how much time they spent in each area was obtained for deployments other than the key rotation. Additional exposures were therefore estimated from the cumulative estimates made for key deployments across all firefighters.
2) To do this their cumulative estimates were divided into attributable portions for each day worked during their secondary deployments. This was done using the estimates calculated on each day in Step 1. A mean exposure for each date worked as a key deployment was obtained. Data was available for almost every day in their deployments (between May 1 st to June 30 th ), however, there were dates, mostly in June, for which no data was available due to the very low or non-existent numbers of respondents initially deployed during those dates. To replace those missing estimates, data from Alberta Environment as a whole was used to calculate the means for each of those days and those were multiplied by 10 hours, approximately the mean number of hours worked for firefighters working in late May and June.
3) This equals a per deployment estimate. These were summed to obtain a cumulative exposure during all non-key deployments and then added to the estimated exposure during the key deployment to estimate total exposure across all deployments.