Characterization and monitoring of biological doses from inhalation exposure to nano and fine particles in industrial workplace

Characterization and monitoring of biological doses from inhalation exposure to nano and fine particles in industrial workplace C. Housiadas, P. Papazafiri 2, P. Neofytou, K. Eleftheriadis, L. Tran 3 ¹ Demokritos, National Centre for Scientific Research, 530 Agia Paraskevi, Athens, Greece 2 School of Biology, University of Athens, 5784, Athens, Greece 3 Institute of Occupational Medicine, Edinburgh, UK

Background Some major steps in the risk assessment sequence: chemical and physical (size distribution) characterization of particles determination of the internally delivered biological doses of the inhaled particles (target tissue doses) biological (toxicological) characterization of particles (response). A study has been undertaken to address all three steps above for the case of industrial manufacturing of detergent and cosmetics (powders). Nationally funded project funded by GSRT (General Secretariat of Research and Technology) Period 20032007

Background (cond.) Relevance to nanoparticles safety SMPSBERNER IMPACTOR 3/07/2005 dm/dlogdp 9.00 Berner impactor 8.00 SMPS, πυκνότητα=.8 g/cc 7.00 6.00 5.00 4.00 3.00 2.00.00.00 0 00 000 0000 Αεροδυναµική διάµετρος, nm Aerodynamic diameter, nm Typically encountered sizes in the fine fraction in the studied cosmetics & detergent production workplace Data from experimental monitoring of airborne exposure levels in a nanoparticle synthesis setting (flame pyrolysis) E. Demou, S. Hellweg, A. Nicolopoulou, D. Mitrakos, C. Housiadas, European Aerosol Conference, Thessaloniki, 2008. E. Demou, L. Tran, P. Neofytou, C. Housiadas, 2 nd International Nanotoxicology Conference, Zurich, 2008

ΑΡΙΘΜ ΤΕΜΑΧ ΑΡΙΘΜ ΑΡΙΘΜΟΣ ΚΙΝΗΤΗΡΩΝ ΤΕΜΑΧ ΑΡΙΘΜ ΑΡΙΘΜΟΣ ΚΙΝΗΤΗΡΩΝ ΤΕΜΑΧ ΑΡΙΘΜΟΣ ΚΙΝΗΤΗΡΩΝ ΙΠΠΟ Υ ΝΑΜΗ(HP) ΙΠΠΟ Υ ΝΑΜΗ(HP) ΙΠ/ΝΑΜΗ ( HP) KW Sampling Detergent & Cosmetics industry in the Athens metropolitan area. Measurements carried out in February 2005 ( week), July 2005 (2 weeks) and November 2006 ( week). OOIIEII: 3 Υ ΡΟΠΛΥΣΤΙΚΟ ΜΗΧΑΝΗΜΑ 2 ΗΛΕΚΤΡΟΚΙΝΗΤΟ ΑΝΥΨΩΤΙΚΟ ΗΛΕΚΤΡΟΚΙΝΗΤΟ ΑΝΥΨΩΤΙΚΟ 2 Α/Α ΟΝΟΜΑΣΙΑ ΠΕΡΙΒΑΛΛΟΝΤΙΚΟΥ ΕΞΟΠΛΙΣΜΟΥ OOIIEII: ΑΕΡΟΚΟΥΡΤΙΝΕΣ Α/Α ΟΝΟΜΑΣΙΑ ΕΞΟΠΛΙΣΜΟΥ ΥΓΙΕΙΝ. & ΑΣΦΑΛΕΙΑΣ ΣΥΝΟΛΟΝ : 208, 42,5 37 ΚΛΕΙΣΤΙΚΗ ΜΗΧΑΝΗ ΧΑΡΤΟΚΙΒΩΤΙΩΝ 36 ΚΛΕΙΣΤΙΚΗ ΜΗΧΑΝΗ ΧΑΡΤΟΚΙΒΩΤΙΩΝ 35 ΚΟΛΛΗΤΙΚΗ ΜΗΧΑΝΗ NOURIS 34 LINX 0,7 3,5 33 ΚΟΛΛΗΤΙΚΗ ΜΗΧΑΝΗ NOURIS 0,7 3,5 32 LINX 3 9 3 30 MICROJET (INKJET) ΖΥΓΙΣΤΙΚΗ ΜΗΧΑΝΗ 32 0 9 8 7 6 5 4 3 2 37 30 29 VIDEOJET (INKJET) 28 ΑΝΤΛΙΑ ΙΑΦΡΑΓΜΑΤΙΚΗ FLUX 27 LINX 26 VIDEOJET (INKJET) 25 MICROJET (INKJET) 2 24 ΜΗΧΑΝΗ WOLF 3 23 ΣΥΣΤΗΜΑ ΙΑΚΙΝΗΣΗΣ ΑΠΟΘΗΚΕΥΣΗΣ SABO 43, 23 22 ΜΗΧΑΝΗ EL PACK 5 29 2 ΓΕΜΙΣΤΙΚΗ ΜΗΧΑΝΗ SENSANI 9 22 20 9 ΜΗΧΑΝΗ ACMA 793 ΜΗΧΑΝΗ ACMA 792,5 6 8 ΚΟΛΛΗΤΙΚΗ ΜΗΧΑΝΗ NOURIS 2 7 ΣΑΚΟΥΛΟΠΟΙΗΤΙΚΗ ΜΗΧΑΝΗ ESSE Gi 7 28 6 5 ΑΝΟΡΘΩΤΙΚΗ ΜΗΧΑΝΗ ΚΙΒΩΤΙΩΝ KEBER ΕΓΚΙΒΩΤΙΣΤΙΚΗ ΜΗΧΑΝΗ ACMA 2 0 2 4 ΚΟΛΛΗΤΙΚΗ ΜΗΧΑΝΗ WEXXAR 0,7 3,5 3 3 2 ΣΥΡΡΙΚΝΩΤΙΚΗ/ΜΑΚΕΤΑΡΙΣΤΙΚΗ ΜΗΧΑΝΗ MAF ΦΟΥΡΝΟΣ 7,5,5 6 ΗΛΕΚΤΡΙΚΟΣ ΠΙΝΑΚΑΣ ΜΗΧΑΝΗΣ MAF 0 ΕΤΤΙΚΕΤΕΖΑ KRONES 8 9 ΠΩΜΑΤΕΖΑ ROLI,5 0000 2 20 8 7 ΓΕΜΙΣΤΙΚΗ ΜΗΧΑΝΗ ROLI ΓΕΜΙΣΤΙΚΗ ΜΗΧΑΝΗ ΜΠΟΥΚΑΛΙΩΝ 6,5 3 6 ΑΝΟΡΘΩΤΙΚΗ ΜΗΧΑΝΗ ΜΠΟΥΚΑΛΙΩΝ POSSIMAT 5,5 27 5 4 ΚΛΕΙΣΤΙΚΗ ΜΗΧΑΝΗ ΚΙΒΩΤΙΩΝ ΣΑΚΟΥΛΟΠΟΙΗΤΙΚΗ ΜΗΧΑΝΗ 3 ΦΟΥΡΝΟΣ,5 6 2 ΣΥΡΡΙΚΝΩΤΙΚΗ/ΜΑΚΕΤΑΡΙΣΤΙΚΗ ΜΗΧΑΝΗ MAF 7,5 33 34 0000 4 5 6 7 8 35 24 3 25 26 ΓΕΜΙΣΤΙΚΗ ΜΗΧΑΝΗ VIONOL MATEER BURT 43 Α/Α ΟΝΟΜΑΣΙΑ ΕΞΟΠΛΙΣΜΟΥ ΗΜΕΡ/ΝΙΑ: ΕΣΧΕ ΙΑΣΘΗ: Γ. ΜΠΑΤΖΑΚΗΣ 9//0 ΑΝΤΙΚΑΘΙΣΤΑ ΤΟ ΣΧ.: 84/R,85 Ground floor filling machine area ΑΝΤΕΓΡΑΦΗ: ΗΛΕΓΧΘΗ: ΚΛΙΜΑΚΑ : :50 ΑΡ. ΓΕΝ. ΣΧΕ ΙΟΥ :. ΑΘΑΝΑΣΙΑ ΗΣ ROLCO ΒΙΑΝΙΛ Α.Ε. 2 30//0 ΙΕΥΘΥΝΣΗ ΤΕΧΝΙΚΗΣ ΥΠΟΣΤΗΡΙΞΗΣ ΑΝΤΙΚ/ΤΑΘΕΙ ΑΠΟ ΤΟ ΣΧ. : ΙΣΟΓΕΙΟ ΣΥΣΚΕΥΑΣΙΑΣ

Methodology (physical characterization) Aerosol measurements with a variety of monitors Examined monitors Reference methods. PDR 000. Passive sampling, based on light scattering, ( wavelength), 00 nm 0 µm 2. PDR 200. Active sampling ( 5 l min), based on light scattering ( wavelength), 00 nm 0 µm 3. DR4000. Active sampling ( 3 l min), based on light scattering (2 wavelengths) 4. Dusttrack. Based on light scattering, may use heads for PM0, PM2.5, PM, 00 nm 0 µm. BERNER impactor, 0 stages between 40 nm 9.3 µm (at 26 l min) 2. SMPS (Model 3080 electrostatic classifier and CPC 3022A), 0 nm µm 3. ANDERSEN impactor, 8 stages between 409 nm 9 µm (at 28.3 l min) 4. GRIMM ENVIROCHECK (model 07) Aerosol Spectrometer, size distribution in the range 0.25 µm 30 µm 5. Sequential particle sampler (FH 95 SEQ), sizes<0 µm, 6 filters 6. PM0, PM2.5 sampling heads (EN234) Objective: examine if measurements made by convenient, inexpensive online instruments (e.g. optical counters) can assess the particulate matter concentration and size characteristics in the workplace

Average workplace exposure monitoring Suitable online instruments to monitor average airborne particle concentration and size characteristics in the workplace: DR4000 + PDR200 mass concentration/logdp µg/m3 20.00 Berner impactor 00.00 PDR2 & DR4 80.00 DR4 60.00 40.00 20.00 0.00 0.0 0. 6/7/05 seq grav, µg/m³ 350.0 300.0 250.0 200.0 50.0 00.0 50.0 0.0 0 20 40 60 80 00 20 40 60 80 pdr200, µg/m³ C PM0 =.09*C PDR200 + 24, R²=0,55 mass concentration/logdp µg/m3 250.00 Berner impactor PDR2 & DR4 200.00 DR4 50.00 00.00 50.00 0.00 3/7/05 0.0 0. 0 Aerodynamic diameter µm

Methodology (chemical characterization) Aerosol particles were collected by a Berner lowpressure impactor: 0 stages between 30 nm 3.35 µm Flow rate 26 l min Tedlar foils Chemical analysis Loaded filters Microwave digestion Ultrasound extraction Electrothermal atomic absorption spectrometry, ETAAS Metals: Cd, Pb, V, Ni, Mn, Cu, Cr, Fe, Al Ion chromatography, IC Watersoluble inorganic ions: SO 4=, NO 3, Cl, HPO 4, NH 4+, Na +, K +, Ca ++

Size distribution Metals Ions C/dlogd ng m 3 200 80 60 40 20 00 80 60 Cu Fe Ni Cr C/dlogd, µg m 3 30 25 20 5 0 Cl NO3 SO4 NH4 Ca 40 20 0 5 0 0.0 0..0 0.0 00.0 size µm 0.0 0. 0 µm unimodal distribution maximum in coarse particles released in the atmosphere during mechanical processes, mixing of raw materials 50 % of the total particle mass consists of SO 4=, NO 3, Cl, NH 4+ and Ca ++ maximum in coarse particles x The watersoluble fraction of fine particles with diameters between 70 nm 850 nm is composed mostly by SO 4= and NH 4 +

DoseResponse Modeling Inhalation Dosimetry Modeling Determine internally delivered biological doses assuming worst case scenarios, i.e. workers are not using protective means Integration of two mathematical models Lung Deposition Lung Clearance D, mechanistic, respiratory deposition modelling including aerosol & breathing dynamics Mitsakou, Helmis, Housiadas, J. Aerosol Sci., 36, 7594, 2005 Mitsakou, Mitrakos, Neofytou, Housiadas, J. Aerosol Medicine, 20, 59529, 2007 Compartmental, mechanistic, modelling of retention/translocation of deposited particles Tran et al., Inhalation Toxicology,, 059076, 999. Tran & Kuempel, in: Particle Toxicology (eds. K. Donaldson, P. Born), 35386, CRC Press, 2007.

Lung deposition Modeling D code with full inhalation dynamics and aerosol dynamics Physical processes Lung morphometry Weibel s Model A (24 generations) trachea bronchi bronchioli Trumpet Model Timevarying crosssectional area

Lung deposition Modeling (cont.) General Dynamic Equation (GDE) temporal variation convection diffusion condensation coagulation 64748 647448 6447448 deposition 6447448 644474448 Ni ( AT N ) i ( AAuN i) ( AT D 678 ) Vd N ( ) ( ) i i AT N = + Γ + i + AT Ni t x x x t growth t coagulation Sectional method: Arbitrary size distribution Particle growth: Mason s equation (Kelvin, solute mass effect, Fuchs correction) Coagulation : Smoluchowski equation (Jacobson et al., 994) Deposition mechanisms: brownian diffusion gravitational settling inertial impaction, all described mechanistically. Equation of continuity A t T = x ( A u) A

Clearance Modeling Compartmental model Schema of the compartments (X to X 9 ) and the rates for transfer of particles between compartments Considered mechanisms: Phagocytosis Macrophage Life Cycle Interstitialization Transfer to the Lymph Nodes Dynamic equation for compartment i dx dt i = k X O ij i ik

Effect of hygroscopicity on lung deposition dm d /(m tot dlogd) 0,00 0,008 0,006 0,004 (NH 4 ) 2 SO 4 CaCl CaCO 2 3 HYGROSCOPIC A Hygroscopic aerosols absorb water Particle mass increases. 0,002 0,000 0, 0 Particle diameter, d (µm) 0,003 (NH 4 ) 2 SO 4 CaCO CaCl 2 3 INERT B Lower deposition if hygroscopicity effects are ignored dm d /(m tot dlogd) 0,002 0,00 0,000 0, 0 Particle diameter, d (µm)

Gravimetric & optical measurements Effect on lung deposition 20 mass concentration/logdp µg/m3 20.00 Berner impactor 00.00 PDR2 & DR4 80.00 DR4 60.00 40.00 20.00 0.00 0.0 0. Particle diameter µm Typical aerosol concentration measurement by Berner impactor and DR4, PDR200 nephelometers dm d /(m tot dlogd ) Berner Dataram4 5 Dataram4&PDR200 0 5 0 0.0 0. 0 00 Particle diameter (µm) Calculated mass deposited on lungs per mass inhaled, as based on measurements by DR4, PDR200 and Berner impactor Deposited Mass in ngr per breath. 6/7/2005 ET TB AI ET: extrathoracic region TB: tracheobronchial region AI: Alveolar region BERNER DR4 DR4 & PDR200 66 4 6 86 268 23 36 490 339

Gravimetric & optical measurements (cont.) Effect on biological dose mass concentration/logdp µg/m3 mass concentration/logdp µg/m3 20.00 Berner impactor 00.00 PDR2 & DR4 80.00 DR4 60.00 40.00 20.00 0.00 0.0 0. 250.00 Berner impactor PDR2 & DR4 200.00 DR4 50.00 00.00 50.00 Alveolar burden ( µg/0 6 epithelial cells) 0.6 0.4 0.2 0. 0.08 0.06 0.04 Monitoring data of 6/7/05 Monitoring data of 3/7/05 0.02 DR4000 + PDR200 Berner 0 0 5 0 5 20 25 30 Time (years) 0.00 0.0 0. 0 Aerodynamic diameter µm Typical aerosol concentration measurement by Berner impactor and DR4, PDR200 Calculated alveolar burden following chronic exposure, as based on measurements by DR4, PDR200 and Berner impactor

DoseResponse Modeling Invitro toxicological assays Toxicological potential of the fractionated material (elution in H 2 O/DMSO) Cell lines A549, MCF7, PC3, A43 MTTa RTPCR Cell viability Expression of following genes: Bcl2, MKP and Hifa

Invitro toxicological assays Cell line: MCF7 Viability:% of control/mass unit Aerosol mass concentration compared to cell Viability over the size range 00 0,00 dc/dlogd 90 80 70 60 50 40 30 20 Aerosol concentration Cell viability 00,00 90,00 80,00 Cell Viability (%) 0 0 70,00 0,00 0,00,000 0,000 00,000 Aerodynamic Diameter The toxicological profile of the collected material was similar in various epithelial cell lines

Size distribution Ions The fraction of fine particles with diameters between 70 850 nm is composed mostly by SO 4= and NH 4 + C/dlogd, µg m 3 30 25 20 5 0 5 Cl NO3 SO4 NH4 Ca 0 0.0 0. 0 00 µm

Invitro toxicological assays (cont.) The water soluble fraction (W) was systematically found to induce higher cytotoxicity than the organic fraction (O) of the total PM0 mass Φίλτρο filter, 24h % % viability επιβίωση 20 0 00 90 80 70 60 50 0 25 50 75 00 µg/ml O W

Invitro toxicological assays (cont.) Expression profile of Hifa with increasing concentrations of eluates from Berner impactor filters CONTROL INCREASING CONCENTRATION Stage 6, (G.M.A.D) = 0.6µm Stage 7, (G.M.A.D) =.2µm B[a]P: 0, 00, 200, 300µM

Preliminary risk assessment ΡΜ0: µg/0 6 cells 0 2.5 5 0 20 viability 00 87.4 74.2 58.9 46.7 DNEL 2.5µg/0 6 epithelial cells Alveolar burden ( µg/0 6 epithelial cells) 0.6 0.4 0.2 0. 0.08 0.06 0.04 Monitoring data of 6/7/05 Monitoring data of 3/7/05 0.02 DR4000 + PDR200 Berner 0 0 5 0 5 20 25 30 Time (years)

Conclusions The dosimetric calculations demonstrate the importance of obtaining the representative measurement of the full size spectrum of the inhaled (nano)aerosol Knowledge (i.e. monitoring) of the size distribution is necessary to assess correctly target doses from inhalation If the deployment of reference monitors is not practical in the industrial setting, the judicious use of simple, inexpensive optical instruments offers a viable alternative to monitor with acceptable accuracy average, sizeresolved workplace exposure levels

Conclusions (cont.) Significantly different lung doses are obtained when particle solubility is taken into account (effect of hygroscopic growth) Knowledge of chemical composition seems necessary to account for hygroscopic effects and assess correctly inhalation dosimetry The presented study illustrates that the feasibility of combining mechanistic mathematical modelling with invitro toxicological assays to derive limits and assess risk

Acknowledgements Demokritos S. Vratolis C. Mitsakou S. Mihaleas A. Zafeiropoulou D. Mitrakos University of Athens O. Mavrofridi M. Xilouri A. Vassilopoulos Technical University of Crete M. Lazaridis T. Glytsos V. Aleksandropoulou J. Ondracek L. Dzumbova University of Crete N. Mihalopoulos P. Zarbas This project is cofunded (75%) by the European Union European Regional Development Fund (ERDF)