Abstract

Most commercial passenger jet aircraft use very hot compressed engine air, after cooling as a source for ventilation and cabin pressurization onboard. This design means that engine oil and/or hydraulic fluid can contaminate the ventilation supply air during otherwise normal flights, exposing onboard crewmembers and passengers to the fumes. The oils and hydraulic fluids contain a complex mixture of triaryl phosphates (TAPs) and decomposition products. Although the health and flight safety consequences of inhaling these fumes have been widely documented, measures of onboard inhalation exposure have been lacking. An approach is presented for documenting exposure to engine oil fumes by using high-resolution mass spectrometry (MS) to monitor and quantify post-translational modifications of subjects’ butyrylcholinesterase (BChE) that are consistent with exposure to the engine oil TAPs. We hypothesized that plasma from exposed individuals would show modifications or adducts of these OPs on the active site serine (Ser198) of BChE. Plasma BChE from 64 exposed subjects was purified to near homogeneity and concentrated using antibodies immobilized on paramagnetic beads. The purified BChE was eluted at low pH, digested with trypsin, and analyzed by liquid chromatography (LC)-MS. In subjects reporting onboard oil fume exposures, the most consistent adduct modifying the Ser198-containing tryptic peptide had a mass value of +154.0031 Da. The normalized peak area (NPA) of the +154Da modification was determined by comparing the relative MS1 intensities of the +154Da-modified Ser198 containing peptide to the total observable peptides containing the active site, including missed cleavages. Notably, adducts from in vitro exposures of bioactivated TAPs to purified BChE conducted in this study (i.e., 80Da, 156Da, 170Da, and 186Da) as well as adducts reported in other earlier in vitro studies (i.e., 65Da, 80Da, 91Da, 107Da, 16Da, 17Da, and 180Da) were not detected in exposed subjects. Of 67 subjects in this study, the average NPA of +154Da-Ser198 resulted from fume event exposures that pre-dated 2012 (N=54; 0.46-17.8, X̅ =4.0) was 7.4X higher than control subjects. These data are uncorrected for the time lag between the reported exposure and the blood draw. Samples from the remaining 13 subjects with exposures from 2016-2024 showed only the 154Da modification at background levels (0.24-1.13; X̅=0.53), as confirmed in control plasma samples from individuals who had not flown in at least three months. The observed reduction in the 154Da adduct over time in exposed individuals is likely a function of the change in the formulation of the OP blends added to engine oils during the course of the study. Further investigation into other protein biomarkers and adducts correlated with exposure to the current oil additives and hydraulic fluid fumes on aircraft is warranted. The most satisfactory solution would be to eliminate the exposure hazard by implementing bleed-free systems or, at a minimum, to develop less toxic oil formulations, suitable bleed air filters, and modified designs.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

We are indebted to the control subjects and oil fume exposed individuals who donated blood to make this research possible. The initial research support was provided by NIEHS (Grant number P42 ES04696) and from the Royal Australian Air Force. More recent funding was provided by cabin crew unions (Association of Flight Attendants-CWA - AFL-CIO; Association of Professional Flight Attendants; British Airlines Stewards and Stewardesses Association - Canadian Union of Public Employees-Airline Division; CUPE Local 4084; International Association of Machinists & Aerospace Workers District Lodge 142 - AFL-CIO - Transport Workers Union - AFL-CIO - Unabhangige Flugbegleiter Organisation - Unite the Union); pilot unions (Aeropers - Air Canada Pilots Association - Allied Pilots Association - Australian Federation of Airline Pilots - Australian and International Pilots Association - Austrian Cockpit Association - Independent Pilots Association - Japan Federation of Aviation Industry Unions - Pilotenverband Swiss Ewiges Wegli - Syndicat National des Pilotes de Ligne - US Airline Pilots Association - Vereiningung Cockpit) - other associations (Australian Licensed Aircraft Engineers Association - Clean Up Cabin Air - Global Cabin Air Quality Executive - International Transport Workers Federation - Sammenslutningen av Fagorganiserte i Energisektoren - Transport and General Workers Union) - and individual aircrew.

Author Declarations

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Human Subjects Committee at the University of Washington gave ethical approval for this study.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Data Availability

All data produced in the present study are avaialable upon reasonable request to the authors.

AbbreviationsBChEbutyrylcholinesterase/plasma cholinesteraseMSmass spectrometryCBDP2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-oneD125Durad 125D150Durad 150DTNB5,5’-dithio-bis-nitrobenzoic aciddd-H2Odouble-distilled waterECHAEuropean Chemicals AgencyNADPHnicotinamide adenine dinucleotide phosphate, reducedOPorganophosphateOPIDNorganophosphate-induced delayed neuropathySDSsafety data sheetSVHCsubstance of very high concernTAPtriaryl phosphateTCPtri-cresyl phosphateHLMshuman liver microsomesTmCPtri-meta-cresyl phosphateTpCPtri-para-cresyl phosphateToCPtri-ortho-cresyl phosphateTMPPtrimethylolpropane phosphateTMPEtri metholylpropane esterTXPtrixylyl phosphate