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Production of well-defined organic fibres for inhalation experiments
J. Aerosol Scl., Yol. 20, No. 8, pp. 1285-1288, 1989. Prlnted in Great Brtta[n.
0021-8502/89 $3.00 + 0.00 Pergamon Press plc
PRODUCTION OF NELL-DEFINED ORGANIC FIBRES FOR INHALATION EXPERIMENTS
J a n M a r t J n i s s e n , Hart Buwalda, Marco van P i n x t e r e n , Saul Lemkowitz, Henk Bibo D e l f t U n i v e r s i t y o f Technology D e l f t F a c u l t y o f Chemical Technology and M a t e r i a l s S c i e n c e S e c t i o n s Risk Management and P a r t i c l e Technology P.O. Box 5045,2600 CA DELFT The N e t h e r l a n d s
Introduction I n view o f the h e a l t h h a z a r d s o f a s b e s t o s f i b r e s , i n d u s t r y i s s e a r c h i n g h a r d f o r s a f e f i b r o u s m a t e r i a l s as s u b s t i t u t e s . An o f t e n quoted t h e o r y s t a t e s t h a t the c a r c i n o g e n t c i t y of fibres i s mainly dependent on t h e i r dimensions (Stanton, 1981), implying t h a t a l l fibrous asbestos s u b s t i t u t e s are dangerous. Stanton°s hypothesis, however, i s n o t based on e x p e r i m e n t s with w e l l - d e f i n e d fibres. We b e l i e v e t h a t b e s i d e s shape o t h e r p a r a m e t e r s , such as c h e m i c a l composition, electrical conductivity and surface structure, contribute s u b s t a n t i a l l y to the h e a l t h h a z a r d s o f f i b r e s . At D e l f t U n i v e r s i t y o f Technology a p r o j e c t i s in p r o g r e s s t o i n v e s t i g a t e the r e l a t i o n between d i f f e r e n t fibre p a r a m e t e r s and t h e i r h e a l t h h a z a r d s . The goals of this investigation a r e to produce w e l l - d e f i n e d f i b r e s o f d i f f e r e n t materials and dimensions and to d i s p e r s e them in a i r in o r d e r to d e t e r m i n e their aerodynamic b e h a v i o u r , p a r t i c u l a r l y in the r e s p i r a t o r y t r a c t . With the information obtained a theoretical model d e s c r i b i n g fundamental aerodynamic behaviour of resptrable f i b r e s w i l l be d e v e l o p e d . Using a l l t h e s e r e s u l t s relevantly sized f i b r e s w i l l be used in c y t o t o x i c o l o g i c a l implantation e x p e r i m e n t s t o d e t e r m i n e f i b r e c a r c i n o g e n i c i t y and maerophage r e a c t i o n . The l i t e r a t u r e r e p o r t s many a t t e m p t s o f r e s e a r c h e r s t o a c h i e v e the production of well-defined fibres (e.g. Spurny e t a l . , 1979). However, sufficient quantities o f more p r e c i s e l y defined f i b r e s are necessary for establishing reliable i n f o r m a t i o n c o n c e r n i n g the aerodynamic b e h a v i o u r and p h y s i o p a t h o l o g i c a l mechanisms o f f i b r e r e s p o n s e . To a c h i e v e t h i s we are d e v e l o p i n g methods o f d i r e c t l y p r o d u c i n g f i b r e s w i t h the d e s i r e d d i m e n s i o n s . Using a number o f t h e s e methods we have a l r e a d y s u c c e e d e d in p r o d u c i n g f i b r e s with h i g h l y c o n s t a n t d i a m e t e r s , down to 0.5 m i c r o m e t e r . ~ethods
v
~polyomid- 6
multilayered stream
Fig. I . The p r o d u c t i o n o f f i b r i l s
gauze
by the s p i n p r o c e s .
1285
fibril
1286
J. MARIJNISSEN et al.
The p r e s e n t e d p r o d u c t i o n method s y n t h e s i z e s m a n - m a d e - o r g a n i c - f i b r e s (MHOF). F i g u r e 1 shows s c h e m a t i c l y t h e p r o d u c t i o n p r o c e s s . Two d i f f e r e n t p o l y m e r s , i n a certain volume r a t i o , flow t h r o u g h a m u l t i f l u x m i x e r (mumi). T h i s m i x e r s p l i t s t h e d o u b l e l a y e r e d p o l y m e r s t r e a m i n t o 2 e q u a l p a r t s and combines them a g a i n i n such a way t h a t 4 p o l y m e r l a y e r s f l o w o u t o f t h e mumi. Using a number o f mumis a m u l t i l a y e r e d s t r u c t u r e i s c r e a t e d , w i t h t h e 2 d i f f e r e n t p o l y m e r s s u c c e e d i n g each other. This m u l t i l a y e r e d stream is forced through a f l u e wire gauze, r e s u l t i n g in the formation of fibrils, as t h e l e s s v i s c o u s p o l y m e r f l o w s a r o u n d t h e more v i s c o u s one. In this way a filament with a diameter of about 40 micrometer is formed, a matrix containing in the order of i00 fibrils of micrometer size diameter. These filaments are stretched, bundled and embedded in a matrix of ice. To obtain fibres of a predetermined length, these bundles are cut to size by a mlcrotome (fig. 2). The ice-matrlx is then removed by melting and the polymer-matrlx by dissolving in a suitable solvent. By adding a stabilizer and diluting with water a suspension of fibres (cuttings of fibrils) is prepared.
~
t I fibril "J
~ilamenf with fibrils ~ ~
~20pm
~ ~ - -~ -}- ' '".... ~rofafion 7cm ~ , .,~%o ~".J . . . . \ ~ microtome
F i g . 2. The embedding and s e c t i o n i n g o f t h e f i l a m e n t s . To n a r r o w t h e v a r i a t i o n i n d i a m e t e r , two methods to s e p a r a t e t h e f i b r e s a c c o r d i n g t o d i a m e t e r can be a p p l i e d . One i s b a s e d on s e d i m e n t a t i o n and t h e o t h e r on i n e r t i a l s e p a r a t i o n ( P r o d i e t a l . , 1982). The r e s u l t i s a s u s p e n s i o n o f h i g h l y m o n o d t s p e r s e f i b r e s , which can be a e r o s o l i z e d by u s i n g a n e b u l i z e r . Results The two p o l y m e r s u s e d i n t h e e x p e r i m e n t s a r e p o l y m e t h y l m e t h a c r y l a t e (PMMA) and p o l y a m i d e - 6 (PA-6 o r n y l o n - 6 ) . Of t h e s e t h e PMMA i s t h e l e s s v i s c o u s , t h u s forming a m a t r i x around nylon f i b r i l s . Several experiments with varying p a r a m e t e r s were p e r f o r m e d i n o r d e r t o f i n d t h e b e s t c o n d i t i o n s t o p r o d u c e f i b r e s with uniform diameters. The b e s t r e s u l t s were o b t a i n e d w i t h t h e f o l l o w i n g c o n d i t i o n s . The v o l u m e - r a t i o PMMA ( t h e l e s s v i s c o u s ) t o n y l o n i s b e t w e e n 6 : 4 and 7:3 t o e n s u r e a good e n c a p s u l a t i o n o f t h e f i b r i l s by t h e m a t r i x . In o r d e r to o b t a i n a good f i b r t l i z a t i o n , t h e combined t h i c k n e s s o f one PMMA l a y e r and one n y l o n l a y e r a t t h e e n t r a n c e o f t h e gauze s h o u l d be l e s s t h a n o r e q u a l t o h a l f t h e gauze a p e r t u r e s i z e . Out o f s e v e r a l p o s s i b i l i t i e s the c o n f i g u r a t i o n with 5 mumis i n s e r i e s f o l l o w e d by t h e gauze and o n l y a s i n g l e f i l a m e n t d r a w i n g d i e , p r o v e d t o g i v e t h e b e s t r e s u l t s so f a r . F i g u r e 3 shows a c r o s s s e c t i o n o f a bundle of filaments. D i s s o l v i n g t h e PMMA i n a c e t o n e y i e l d s f r e e n y l o n f i b r i l s (fig. 4).
Production of well-deflned organic fibres
1287
Fig. 3. Cross section of produced filaments. The white dots are the nylon fibrils, which are surrounded by the greyish PMMA matrix. Filament diameter is about 40 pm, fibril diameter is around i ~m.
Fig. 4. Liberated nylon fibrils, with diameters
in the order of 1 #m.
Some hundred filaments are bundled and embedded in water, which is frozen. These bundles are cut with a Reichert-Jung Frigocut 2700 rotation microtome into sections of 20 micron thickness. The ice is removed by melting and the PMMA by adding acetone. So far no stabilizer is added to keep the fibres in suspension. To measure the dimensions of the fibres use has been made of a scanning electron mlcrograph and a Joyce Loebl Magiscan 2A image analyser. Figure 5 gives a scanning electron micrograph of a sample of cut fibres in suspension. The lengths of the fibres in a sample have an average of 16.4 micron and a relative standard deviation of 22 Z. In the same sample the relative standard deviation of the diameters, as determined from the electron mlcrographs, exceeds I00 %. To increase uniformity in diameter a separation method based on
sedimentation
has been a p p l i e d .
One o f the produced s i z e c l a s s e s i s d e p i c t e d in
figure 6. The diameters have an average of 0.94 micron and the relative standard deviation is 21 Z.
1288
J. MARIJNISSEN et al.
4;
Fig 5. Cut fibres after dissolving the PMMA, before separation by sedimentation. L = 20 ~m, D = 0.i - 4 ~m.
Fig 6. Fibres, a size interval as selected by sedimentation. D = 0.94 ~m.
Conclusion and discussion It is clear that, using the described spin process combined with cutting and size separation according to diameter, monodisperse fibres can be produced. Modelling the production process reveals the most important parameters and it is envisaged that already during the spin process reasonablely uniform diameters can be produced. Although until now only fibres of 20 micrometer have been cut, no problems are anticipated in cutting down till i0 or 5 micrometer. Separation based on sedimentation narrows the range in diameter size. Another method, based on inertial separation (Prodl et al., 1982) will be tested to compare results. Tests are already in progress in which during the production process fluorescent material is added to the nylon, in order to make it possible to trace the fibres during transport and after deposition in the lung model. For this purposes two alternatives are available: radioactively labeled fibres and Laser-Doppler anemometry. Finally it is envisaged to treat the fibres such that their surface properties and electrical behavlour are changed. In this way the factors influencing the transport and deposition behaviour of fibres in lung models can be studied individually. References. Spurny, K.R., Stober, W., Oplela, H., Weiss, G., Size-selective preparation of inorganic fibres for biological experiments, Am. Ind. Hyg. Assoc. J. 40 (1979), 20-38. Stanton, M.F., Layard, M., Tegerls, A., Miller, E., May, M., Morgan, E., Smith, A., The relation of particle dimension to carclnogeneclty in amphibole asbestoses and other fibrous materials, J. Nat. Canc. Inst. 67 (1981), 965-975. van Pinxteren, M., Preparation of well defined micron-range fibres, M.Sc. thesis, Faculty of Chemical Technology and Materials Science, Delft University of Technology, Delft, 1988. Prodl, V., De Zalacomo, T., Fibre collection and measurement with the inertial spectrometer, J. Aerosol Scl. 13 (1982), 49-58.
0021-8502/89 $3.00 + 0.00 Pergamon Press plc
PRODUCTION OF NELL-DEFINED ORGANIC FIBRES FOR INHALATION EXPERIMENTS
J a n M a r t J n i s s e n , Hart Buwalda, Marco van P i n x t e r e n , Saul Lemkowitz, Henk Bibo D e l f t U n i v e r s i t y o f Technology D e l f t F a c u l t y o f Chemical Technology and M a t e r i a l s S c i e n c e S e c t i o n s Risk Management and P a r t i c l e Technology P.O. Box 5045,2600 CA DELFT The N e t h e r l a n d s
Introduction I n view o f the h e a l t h h a z a r d s o f a s b e s t o s f i b r e s , i n d u s t r y i s s e a r c h i n g h a r d f o r s a f e f i b r o u s m a t e r i a l s as s u b s t i t u t e s . An o f t e n quoted t h e o r y s t a t e s t h a t the c a r c i n o g e n t c i t y of fibres i s mainly dependent on t h e i r dimensions (Stanton, 1981), implying t h a t a l l fibrous asbestos s u b s t i t u t e s are dangerous. Stanton°s hypothesis, however, i s n o t based on e x p e r i m e n t s with w e l l - d e f i n e d fibres. We b e l i e v e t h a t b e s i d e s shape o t h e r p a r a m e t e r s , such as c h e m i c a l composition, electrical conductivity and surface structure, contribute s u b s t a n t i a l l y to the h e a l t h h a z a r d s o f f i b r e s . At D e l f t U n i v e r s i t y o f Technology a p r o j e c t i s in p r o g r e s s t o i n v e s t i g a t e the r e l a t i o n between d i f f e r e n t fibre p a r a m e t e r s and t h e i r h e a l t h h a z a r d s . The goals of this investigation a r e to produce w e l l - d e f i n e d f i b r e s o f d i f f e r e n t materials and dimensions and to d i s p e r s e them in a i r in o r d e r to d e t e r m i n e their aerodynamic b e h a v i o u r , p a r t i c u l a r l y in the r e s p i r a t o r y t r a c t . With the information obtained a theoretical model d e s c r i b i n g fundamental aerodynamic behaviour of resptrable f i b r e s w i l l be d e v e l o p e d . Using a l l t h e s e r e s u l t s relevantly sized f i b r e s w i l l be used in c y t o t o x i c o l o g i c a l implantation e x p e r i m e n t s t o d e t e r m i n e f i b r e c a r c i n o g e n i c i t y and maerophage r e a c t i o n . The l i t e r a t u r e r e p o r t s many a t t e m p t s o f r e s e a r c h e r s t o a c h i e v e the production of well-defined fibres (e.g. Spurny e t a l . , 1979). However, sufficient quantities o f more p r e c i s e l y defined f i b r e s are necessary for establishing reliable i n f o r m a t i o n c o n c e r n i n g the aerodynamic b e h a v i o u r and p h y s i o p a t h o l o g i c a l mechanisms o f f i b r e r e s p o n s e . To a c h i e v e t h i s we are d e v e l o p i n g methods o f d i r e c t l y p r o d u c i n g f i b r e s w i t h the d e s i r e d d i m e n s i o n s . Using a number o f t h e s e methods we have a l r e a d y s u c c e e d e d in p r o d u c i n g f i b r e s with h i g h l y c o n s t a n t d i a m e t e r s , down to 0.5 m i c r o m e t e r . ~ethods
v
~polyomid- 6
multilayered stream
Fig. I . The p r o d u c t i o n o f f i b r i l s
gauze
by the s p i n p r o c e s .
1285
fibril
1286
J. MARIJNISSEN et al.
The p r e s e n t e d p r o d u c t i o n method s y n t h e s i z e s m a n - m a d e - o r g a n i c - f i b r e s (MHOF). F i g u r e 1 shows s c h e m a t i c l y t h e p r o d u c t i o n p r o c e s s . Two d i f f e r e n t p o l y m e r s , i n a certain volume r a t i o , flow t h r o u g h a m u l t i f l u x m i x e r (mumi). T h i s m i x e r s p l i t s t h e d o u b l e l a y e r e d p o l y m e r s t r e a m i n t o 2 e q u a l p a r t s and combines them a g a i n i n such a way t h a t 4 p o l y m e r l a y e r s f l o w o u t o f t h e mumi. Using a number o f mumis a m u l t i l a y e r e d s t r u c t u r e i s c r e a t e d , w i t h t h e 2 d i f f e r e n t p o l y m e r s s u c c e e d i n g each other. This m u l t i l a y e r e d stream is forced through a f l u e wire gauze, r e s u l t i n g in the formation of fibrils, as t h e l e s s v i s c o u s p o l y m e r f l o w s a r o u n d t h e more v i s c o u s one. In this way a filament with a diameter of about 40 micrometer is formed, a matrix containing in the order of i00 fibrils of micrometer size diameter. These filaments are stretched, bundled and embedded in a matrix of ice. To obtain fibres of a predetermined length, these bundles are cut to size by a mlcrotome (fig. 2). The ice-matrlx is then removed by melting and the polymer-matrlx by dissolving in a suitable solvent. By adding a stabilizer and diluting with water a suspension of fibres (cuttings of fibrils) is prepared.
~
t I fibril "J
~ilamenf with fibrils ~ ~
~20pm
~ ~ - -~ -}- ' '".... ~rofafion 7cm ~ , .,~%o ~".J . . . . \ ~ microtome
F i g . 2. The embedding and s e c t i o n i n g o f t h e f i l a m e n t s . To n a r r o w t h e v a r i a t i o n i n d i a m e t e r , two methods to s e p a r a t e t h e f i b r e s a c c o r d i n g t o d i a m e t e r can be a p p l i e d . One i s b a s e d on s e d i m e n t a t i o n and t h e o t h e r on i n e r t i a l s e p a r a t i o n ( P r o d i e t a l . , 1982). The r e s u l t i s a s u s p e n s i o n o f h i g h l y m o n o d t s p e r s e f i b r e s , which can be a e r o s o l i z e d by u s i n g a n e b u l i z e r . Results The two p o l y m e r s u s e d i n t h e e x p e r i m e n t s a r e p o l y m e t h y l m e t h a c r y l a t e (PMMA) and p o l y a m i d e - 6 (PA-6 o r n y l o n - 6 ) . Of t h e s e t h e PMMA i s t h e l e s s v i s c o u s , t h u s forming a m a t r i x around nylon f i b r i l s . Several experiments with varying p a r a m e t e r s were p e r f o r m e d i n o r d e r t o f i n d t h e b e s t c o n d i t i o n s t o p r o d u c e f i b r e s with uniform diameters. The b e s t r e s u l t s were o b t a i n e d w i t h t h e f o l l o w i n g c o n d i t i o n s . The v o l u m e - r a t i o PMMA ( t h e l e s s v i s c o u s ) t o n y l o n i s b e t w e e n 6 : 4 and 7:3 t o e n s u r e a good e n c a p s u l a t i o n o f t h e f i b r i l s by t h e m a t r i x . In o r d e r to o b t a i n a good f i b r t l i z a t i o n , t h e combined t h i c k n e s s o f one PMMA l a y e r and one n y l o n l a y e r a t t h e e n t r a n c e o f t h e gauze s h o u l d be l e s s t h a n o r e q u a l t o h a l f t h e gauze a p e r t u r e s i z e . Out o f s e v e r a l p o s s i b i l i t i e s the c o n f i g u r a t i o n with 5 mumis i n s e r i e s f o l l o w e d by t h e gauze and o n l y a s i n g l e f i l a m e n t d r a w i n g d i e , p r o v e d t o g i v e t h e b e s t r e s u l t s so f a r . F i g u r e 3 shows a c r o s s s e c t i o n o f a bundle of filaments. D i s s o l v i n g t h e PMMA i n a c e t o n e y i e l d s f r e e n y l o n f i b r i l s (fig. 4).
Production of well-deflned organic fibres
1287
Fig. 3. Cross section of produced filaments. The white dots are the nylon fibrils, which are surrounded by the greyish PMMA matrix. Filament diameter is about 40 pm, fibril diameter is around i ~m.
Fig. 4. Liberated nylon fibrils, with diameters
in the order of 1 #m.
Some hundred filaments are bundled and embedded in water, which is frozen. These bundles are cut with a Reichert-Jung Frigocut 2700 rotation microtome into sections of 20 micron thickness. The ice is removed by melting and the PMMA by adding acetone. So far no stabilizer is added to keep the fibres in suspension. To measure the dimensions of the fibres use has been made of a scanning electron mlcrograph and a Joyce Loebl Magiscan 2A image analyser. Figure 5 gives a scanning electron micrograph of a sample of cut fibres in suspension. The lengths of the fibres in a sample have an average of 16.4 micron and a relative standard deviation of 22 Z. In the same sample the relative standard deviation of the diameters, as determined from the electron mlcrographs, exceeds I00 %. To increase uniformity in diameter a separation method based on
sedimentation
has been a p p l i e d .
One o f the produced s i z e c l a s s e s i s d e p i c t e d in
figure 6. The diameters have an average of 0.94 micron and the relative standard deviation is 21 Z.
1288
J. MARIJNISSEN et al.
4;
Fig 5. Cut fibres after dissolving the PMMA, before separation by sedimentation. L = 20 ~m, D = 0.i - 4 ~m.
Fig 6. Fibres, a size interval as selected by sedimentation. D = 0.94 ~m.
Conclusion and discussion It is clear that, using the described spin process combined with cutting and size separation according to diameter, monodisperse fibres can be produced. Modelling the production process reveals the most important parameters and it is envisaged that already during the spin process reasonablely uniform diameters can be produced. Although until now only fibres of 20 micrometer have been cut, no problems are anticipated in cutting down till i0 or 5 micrometer. Separation based on sedimentation narrows the range in diameter size. Another method, based on inertial separation (Prodl et al., 1982) will be tested to compare results. Tests are already in progress in which during the production process fluorescent material is added to the nylon, in order to make it possible to trace the fibres during transport and after deposition in the lung model. For this purposes two alternatives are available: radioactively labeled fibres and Laser-Doppler anemometry. Finally it is envisaged to treat the fibres such that their surface properties and electrical behavlour are changed. In this way the factors influencing the transport and deposition behaviour of fibres in lung models can be studied individually. References. Spurny, K.R., Stober, W., Oplela, H., Weiss, G., Size-selective preparation of inorganic fibres for biological experiments, Am. Ind. Hyg. Assoc. J. 40 (1979), 20-38. Stanton, M.F., Layard, M., Tegerls, A., Miller, E., May, M., Morgan, E., Smith, A., The relation of particle dimension to carclnogeneclty in amphibole asbestoses and other fibrous materials, J. Nat. Canc. Inst. 67 (1981), 965-975. van Pinxteren, M., Preparation of well defined micron-range fibres, M.Sc. thesis, Faculty of Chemical Technology and Materials Science, Delft University of Technology, Delft, 1988. Prodl, V., De Zalacomo, T., Fibre collection and measurement with the inertial spectrometer, J. Aerosol Scl. 13 (1982), 49-58.