A Field Test of a Procedure for the Identification of Protein-Bearing

16 A Field Test of a Procedure for the Identification of

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Protein-Bearing Particles in Grain Elevator A i r R. M. BUCHAN and G. D. KRAMER Occupational Health and Safety Section, Colorado State University, Fort Collins, CO 80523 Grain elevators are a major component of an extensive agricultural distribution system. Over 100,000 people are employed at the hundreds of large elevators and the thousands of small country elevators located throughout the United States1. These workers are exposed to considerable amounts of grain dust which is generated each time grain i s handled. Although grain dust i s considered as a nuisance particulate2, and as such is supposed to be "biologically inert", there have been numerous studies which have documented that exposure to grain dust resulted in the development of pulmonary disease in a large number of workers. The f i r s t report of respiratory difficulty in grain handlers appeared in 1713 when Ramazzini3 observed that workers in graineries and barns, engaged in sifting and measuring grain, almost all developed shortness of breath and rarely reached old age. Later epidemiological and clinical investigations of grain handlers have documented a variety of symptoms which resulted from exposure to grain dust. The most commonly reported symptoms in these studies included chronic cough, dyspnea, tightness across the chest and grain fever. In addition, reduced pulmonary function and pulmonary fibrosis were frequently noted4. Although the association between the development of pulmonary disease and exposure to grain dust has been recognized for a long period of time the mechanisms by which grain dust exerts i t s harmful effects remains largely unknown. A number of authors have suggested that the development of pulmonary disease may be primarily the result of a foreign protein reaction. A survey of the literature indicated that the specific reactions involved may be Type 1 and Type III hypersensitivity reactions3-7. Since the induction of pulmonary disease may be the result of a foreign protein reaction, rather than measuring a workers total dust exposure as is presently done, it may be more advantageous to examine the component of that exposure that consists of protein bearing particles. The purpose of this study was to demonstrate a method for determining particulate protein concentrations and 0-8412-0539-6/80/47-120-301$05.00/0 © 1980 American Chemical Society In Analytical Techniques in Occupational Health Chemistry; Dollberg, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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size distribution i n grain elevator atmospheres and to compare these parameters with those found under ambient conditions.


Methods and Materials Sampling Methodology. A i r samples were taken at four grain elevator work sites and one s i t e outside to serve as a control. The sampling areas were as follows: a) Site 1 was located on the top floor of a country elevator where grain was loaded i n 24 storage bins; b) Site 2 was located on the main work floor of the county elevator where most of the elevator functions were directed from; c) Site 3 was located on the bottom floor of the county elevator where grain was transported from the bottom of the storage bins to the loading area; d) Site 4 was located on the main work floor of the adjacent manufacturing elevator, and e) Site 5, which served as the control, was located approxi mately 250 yards upwind of the two grain elevators. Due to high dust concentrations inside the elevators, samples were collected with Dupont low flow sampling pumps (model P125) operated at a flow rate of approximately 120 cc/min. The outside control samples were collected using a Bendix Sequential A i r Sampler modified for membrane f i l t r a t i o n and operated a t a flew rate of approximately 6 lpm due to low particulate concentrations of the ambient atmosphere. A l l of the samples were collected on Millipore Type HA f i l t e r s which have a pore size of 0.45 ym. Particle Staining Procedure. As soon as possible after the samples were collected they were strained for protein according to a procedure developed by Magill and Lumpkins . The reagents used i n the procedure were as follows: Reagent A - 1% by weight aqueous n i t r i c acid Reagent Β - 1% aqueous solution Ninhydrin i n water Reagent C - 0.2% Wool Fast Pink RL i n 10% acetic acid solution Reagent D - 95% undenatured ethyl alcohol The staining procedure involved placing an absorbent paper pad into four p e t r i dishes and adding just sufficient amounts of reagents A, B, C, and D to saturate the pad without immersing i t . A representative section (one fourth wedge) of the Millipore f i l t e r , with dust deposition side up, was placed on each of the pads for two minutes. Between each treatment the bottom side of the f i l t e r was blotted on a paper towel to remove excess solution. After the f i n a l treatment the f i l t e r was dried for one hour a t roan temperature. The dry, stained f i l t e r was then placed on a clear microscope slide and made translucent with immersion o i l . Permanent slides were made by sealing the coverslips to the slides with clear fingernail polish. The mechanism involved i n the staining procedure are not 6

In Analytical Techniques in Occupational Health Chemistry; Dollberg, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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Proteins

in Grain Elevator

Air

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completely understood. Ninhydrin i s frequently used to stain proteinaceous matter but i t s role i n the procedure developed by Magill and Lumpkins^ remains uncertain. I t has been suggested that Ninhydrin may react with some charged groups on the protein molecule and thus enhance dye binding. This i s unlikely, however since the pH of the solutions (1.3 - 3.0) should prohibit the Ninhydrin reaction. Following the f i e l d investigation which u t i l i z e d this staining technique, an attempt was made to determine i f Ninhydrin was indeed a necessary reagent. Thus a test was run i n which one half of a dust sample was treated according to the procedure described by Magill and Lumpkins§, while the other half of the f i l t e r was treated identically except the Ninhydrin step was emitted. The resultant stain appeared to be of equal intens i t y on both f i l t e r halves. Therefore, i t was l i k e l y that Ninhydrin has neither a positive nor a negative effect on the staining. The mechanisms involved i n the binding of Wool Fast Pink RL (Figure 1) to the protein molecule i s as well not f u l l y understood but probably involves a combination of ionic interactions be-bween the charged groups on the dye and protein molescules, and hydrophobic interactions between the dye and protein molecules. Particle Analysis. When evaluating a workers exposure by determining size count distribution data for dust collected on membrane f i l t e r s and analysis by l i g h t microscopy, the particles must be sized i n a manner which w i l l r e f l e c t geometric, aerodynamic, or biological properties to a s u f f i c i e n t l y close degree to be an accurate representation of particle properties related to size. In this study sizing was accomplished by determining the projected area of the particle employing the graduated c i r c l e s of a Porton reticule. The counting technique was based on the truncated multiple traverse technique as described by Sichel^. This i s a s t a t i s t i c a l l y based method of selecting and counting f i e l d s i n a s t r a t i f i e d manner which improves r e l i a b i l i t y , ntinimizes the

C . I . A c i d Red

Figure 1.

289

(£Ui ish

pink —>bright

bluish

red)

Suggested structural formula for Wool Fast Pink RL



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number of individual measurements and i s more s t a t i s t i c a l l y unbiased. The f i r s t step i n the procedure requires a traverse of the f i l t e r beginning a t the center and progressing to the outside edge. A t o t a l of ten f i e l d s per f i l t e r were randomly chosen per traverse. During this and each subsequent traverse of the f i l t e r , the number of particles i n each particle size interval was recorded. Following the completion of 10 traverses the number of particles i n each size interval was totaled and weighted by dividing the t o t a l number of particles i n that size interval by the number of traverses made to achieve that t o t a l . By this method the number of particles per traverse was calculated. The average number of particles i n each size interval was progressively cumulated and the cumulated percentage of particles i n each size interval was then calculated for making plots, size versus normalized cumulative percent on log probability graph paper. Data Analysis. When the cumulative percentage of particles i n each size interval was plotted against particle diameter on log probablity paper the data points approximated a straight line. The regression l i n e through the experimental data points was then calculated by the least squares method. Estimates o f the geometric median diameter, the geometric standard deviation, and the percentage of the distribution composed of respirable (£ 10 ym diameter) particulates were calculated from the regression equation. In addition, the concentration, and percentage of the concentration composed of protein bearing particles, were calculated. These five parameters were then subjected to an analysis of variance procedure to determine i f they differed significantly between sites. A difference was considered s i g n i ficant i f the p-value was less than or equal to the 0.05 level. Results and Discussion The staining of protein bearing particles by the method described by Magill and Lumpkins^, coupled with size-count analysis by l i g h t microscopy, proved to be an excellent means of measuring the component of a workers exposure composed of protein bearing particulates. The stained, protein bearing particles could be easily distinguished f ran non-protein bearing particles and were clearly v i s i b l e i n sizes as small as one micraneter i n diameter. The f i v e parameters which were analyzed for protein bearing and t o t a l dust distributions a t each sampling s i t e were the geometric median diameter, the geanetric standard deviation, the percentage of particles which were respirable U 10 ym diameter), the concentration, and the percent of the t o t a l concentration composed of protein bearing particles. The results of the s t a t i s t i c a l analysis are summarized i n Table I. As can be seen i n Table I, the geanetric median diameter was not significantly different f o r protein bearing particles between the elevator



-

*Millions of particles per cubic meter of a i r ym = micrometers

-

-

38.0%

6.1%

Yes

9.2

Yes

0.31

44.5

Concentration*

Percent of Distribution Containing Protein

Yes

99.6

98.5%

Yes

95.4%

97.2%

Per Cent Pespirable

123.8

No Yes

6.12

4.50

Yes

P*0.05

No

0.10 ym

Ambient

6.89

0.53 ym

Elevator

5.40

No

PsO.05

Geometric Standard Deviation

0.45 ym

Ambient

0.47 ym

Elevator

Total Dust

Geometric Median Diameter

Parameter

Protein Bearing Dust

A Comparison of Mean Values of the Protein Bearing and Total Dust Distribution Parameters Between Elevator and Ambient A i r

TABLE I



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sites and the control s i t e . If i t i s the protein bearing dust which i s primarily responsible for the induction of pulmonary disease, then dust exposures between the elevator sites and the control sites should not be considered different, i n terms of the geanetric mean diameter since p a r t i ζ le size of both distributions are similar and thus would have l i k e characteris t i c s for pulmonary deposition The geanetric standard deviation was not significantly different for the protein bearing or t o t a l dusts between the elevator sites and the control s i t e . Since the geanetric standard deviation and the geanetric median diameter for protein bearing particles did not d i f f e r significantly between elevator sites and the control s i t e , dust exposures at these sites should not be considered differently i n terms of these two parameters. They may, however, d i f f e r significantly i n terms of other parameters such as concentration or percent protein. The fraction of the dust distribution composed of respirable particles did d i f f e r significantly bet**een the elevator sites and the control s i t e for both protein bearing and t o t a l particles. However, since the percent of the distribution canposed of respirable particles was so large, and averaged 97.2% for the protein bearing particles and 98.5% for the t o t a l particles, i t i s doubtful that these differences would be biologically s i g n i f i cant fran a health standpoint as t o t a l dose i n terms of numbers of inhaled particles would be only s l i g h t l y different. The concentration of both protein bearing and t o t a l dust were markedly higher at the elevator sites than at the control s i t e . I t i s obvious that the higher conœntrations at the elevator sites would present a greater health hazard than the concentration at the control s i t e . In addition, the percent of the concentrations canposed of protein bearing particles ranged fran 35.0% to 44.7% at the elevator sites and was only 6.1% at the control s i t e . Thus, an evaluation of a worker's exposure based on t o t a l dust alone may not r e f l e c t a l l ramifications of the true hazard potential, since this would not r e f l e c t the relative percentage of protein bearing particles between the elevator sites and the control sites. I t i s apparent that measurement of the protein bearing canponent of a dust sample may be a more accurate means of assessing the health potential associated with dust exposures i n grain elevators. In the future, this method of measuring the canponent of a worker's exposure which i s made up of protein bearing particles could be used to investigate the œrrelation between protein bearing dust exposures and pulmonary signs and symptans among grain handlers. If a good œrrelation were found, i t might contribute to a clearer understanding of the etiology of grain dust induced lung disease.


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Literature Cited 1.

Lehmann, P.: "Grain Elevator Hazards," Job Safety and Health,

2.

Threshold Limit Values for Chemical Substances and Physical Agents in the Workroom Environment. American Conference of Governmental Industrial Hygienists, Cincinnati, Ohio, 1978. Ramazzini, B.: Diseases of Workers. Translated by W. C. Wright. Hafner Publishing Company, New York (1964). DoPico, G. Α., et. a l : "Respiratory Abnormalities Among Grain Handlers," American Review of Respiratory Disease,


3. 4. 5. 6.

3:4-9

(1975).

115:915-927

(1978).

Cohen, V. L. and H. Osgood: "Disability Due to Inhalation of Grain Dust," J. Allergy, 24:193-211 (1953). Tse, K. S., P. Warren, M. Janusz, D. S. McCartney and R. M. Cherniack: "Respiratory Abnormalities in Workers Exposed to Grain Dust," Archives of Environmental Health, 27:74-77 (1973).

7.

Warren, P., R. M. Cherniack and K. S. Tse: "Hypersensi tivity Reactions to Grain Dust, "J. Allergy and Clinical

8.

Magill, P. L. and E. D. Lumpkins: "Distinguishing Skin Scale Particles," Contamination Control, October, 1966. Sichel, H. S.: "On the Size Distribution of Airborne Mine Dust," J. South African Institute of Mining and Metalurgy,

9.

Immunology, 53:139-149 (1974).

58:171-225

(1957).

RECEIVED October 17, 1979.


A Field Test of a Procedure for the Identification of Protein-Bearing

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