The value of microscopy analysis Garry Burdett Health and Safety Laboratory Harpur Hill, Buxton, UK, SK17 9JN

Asbestos Hazard Mortality (excess deaths) due to: –Fibrosis of the lung (asbestosis) –Cancer of the lung (bronchial cancer etc) –Cancer of the lung lining (e.g. mesothelioma) Morbidity (lung changes/dysfunction) –Pleural plaques; –Diffuse pleural effusions

Fibre Related Risk: Depends on: –Dimensions –Durability / Type –Dose / Exposure –Time (Measurable by microscopy)

EU Directive: Dimensions For the purposes of measuring in the air, only fibres with a length of more than five micrometres, a breadth of less than three micrometres and a length to breadth ratio greater than 3:1 shall be taken into consideration. (2003/18/EC amending to EU asbestos worker protection directive (83/477/EEC) article 7.6 )

Durability / Type

Dose / Exposure Exposure is used as a surrogate for dose by sampling a known volume of air onto a membrane filter and counting the numbers of fibres present in a known area. This is used to calculate the fibre concentration in f/ml or f/cm 3 of air. Uses size criteria to determine ability to reach pulmonary region of the lung.

Time Dose = exposure x duration (f/ml/years) Risk  cumulative dose (assume linear); Risk  time since first exposure (mesothelioma related by power law). Lag time 15 – 60 years from exposure to disease symptoms

EU directive: Fibre Counting “Fibre counting shall be carried out whenever possible by PCM (Phase contrast microscope) in accordance with the 1997 (World Health Organisation) recommended method (16) or any other method giving equivalent results.”

EU Directive: Requirements The 2003 amendment to 83/477/EEC abandoned action levels and different control levels for different types of asbestos and introduced a single limit of 0.1 f/ml over an 8-hour time weighted average. (Article 8) Therefore the EU regulation for controlling the risk to workers, require only PCM fibre number information over a broadly defined size range. Article 4 requires the type and quantities of asbestos handled to be notified

Main types of microscopy for fibre counting. Phase contrast light microscopy PCM Scanning electron microscopy SEM Transmission electron microscopy TEM. All have ISO or WHO standard methods Polarised light microscopy PLM for bulk analysis.

PCM (X500 magnification)

SEM

TEM

SEM higher Mag & 3D

TEM high mag chrystotile

PLM : Dispersion staining

Discrimination of fibre type PCM: No discrimination or identification ; PLM Uses optical properties + refractive index/ dispersion colours to identify fibres; >0.8µm width; SEM uses Energy Dispersive X-ray analysis to semi-quantitatively measure chemistry; TEM uses quantitative EDX and electron diffraction to identify fibres.

Comparison of methods for fibre counting Operating Mag Area (mm 2 ) examined Time taken (mins.) Rate area examined mm 2 /hr PCMX500(200 fov) SEMX2000(50 fov) TEMx5000(50 go)

Likely upper limit of detection (f/ml) by method and volume WORKENVIRON MENTAL COST PER HOUR VOLUME (litres) PCM0.01*0.0017*30-50 SEM TEM

Accuracy & Precision Accuracy not usually known. Precision includes: –Random “Poisson” counting error –Instrumental errors (QA / calibration) –Human errors (QA / Proficiency testing)

Random counting errors (95%) No. of fibres Lower limit Upper limit No. of fibres Lower limit Upper limit

Quality and accreditation Quality assurance and proficiency testing is essential, particularly if results from different analysts and laboratories are being used or compared. Accreditation of Laboratories to ISO mandated by the EU directive.

Type of analysis PCMSEMTEMPLM Fibre counting of air samples Belgium (25) France (?) Netherlands(2 0) Spain (20) UK (200) Germany* France (20)Not applicable Fibre Identification of bulk samples Not applicableUK (5)UK (17)UK (250) Fibre counting and identification of air samples Not applicableGermany* France (20)Not applicable * PT scheme run for one or two rounds in past a new scheme was planned Note: UK schemes also include a number of EU countries who participate

RICE: Fibre counting PT RICE labs. MMMF – 12 labs. ERM – WHO major change

AIMS: Fibre Identification PT

Value of PCM counting 1.Workplace exposure can be controlled using the WHO - PCM method; 2.Counts “regulated” WHO fibres & meets EU criteria; 3.Relatively quick (on-site) and cheap; 4.Environmental exposure can be screened by PCM.

PCM Limitations 1.PCM has limited detection due to background counts on blank filters (0.01 f/ml); 2.No knowledge of fibre types; 3.Only an “index of exposure”

Value of analytical SEM Can be adjusted to give regulated fibre counts, Can give fibre size information, Classifies fibre types present using EDX spectra data.

SEM limitations Limited availability; Off site analysis; Costly Can only be used for regulated fibre counting, No definitive identification of fibres, Lowest area of filter scanned per field of view.

Value of TEM Can count regulated fibres + all fibre sizes. Accurate size information, EDX chemical analysis of all fibre sizes Crystal structure information (SAED) Identification of fibre type, Can give complete information on all fibre sizes if needed

Limitations of TEM Limited availability; Off site; Costly

Recommendations 1 Use PCM for workplace control and checking after clean –up (with disturbance sampling). Indoor and near source environmental use PCM screening and SEM or TEM analysis (as necessary) on selected half-filters. Ambient air: Analytical TEM is best, SEM is more limited, PCM not very useful.

Recommendations 2 Use Polarised light microscopy (PLM) for analysis of bulk samples for asbestos present and type of asbestos. Use PLM to screen minerals and soils for asbestos content. Asbestos in water usually analysed by TEM