Identification of a particular amphibole asbestos fiber in tissues of persons exposed to a high oral intake of the mineral

ENVIRONMFi%TaL

RESEARCH

21. 85~93

(1980)

Identification of a Particular Amphibole Asbestos Fiber in Tissues of Persons Exposed to a High Oral intake of the Mineral

Samples of liver. jejunum, and lung tissue from each of 32 persons with long-term, highlevel oral exposure to a ferromagnesium silicate amphibole asbestos were ashed, and the residue was analyzed by means of transmission electron microscopy with selected area electron diffraction. and energy dispersive X-ray analytic techniques. Fibers of the type ingested were identified in the tissues of 29 of the 32 persons. Among ?I controls, the tissues of only 2 subjects contained this type of fiber-in each. a single fiber from a single tissue specimen. Chrysotile asbestos fibers were found in all tissues in most of the subjects studied.

INTRODUCTION Serpentine (chrysotile) and many amphibole minerals occur in fibrous habit and are widely distributed in nature. They are found in potable water supplies and beverages (Cunningham and Pontefract, 1971, 1973: Nicholson and Pundsack, 1973: Cook t’t (I/.. 1974: Nicholson, 1974). Commercial use of these minerals (95% chrysotile) is widespread and is increasing, resulting in considerable exposure for some industrial workers and in small. inadvertent exposures for large population groups not directly involved in industrial contact. With few exceptions, exposures have been to airborne fibers, coming from such diverse sources as automobile brake linings or the asbestos-concrete mixtures sprayed on structural girders in steel-frame buildings. Chrysotile fibers have been identified in the lung tissue of urban dwellers who have had no apparent industrial contact (Langer et (II.. 1971). The biologic hazard of inhaled fibrous silicates has been extensively documented (Selikoff rt trl.. 1964: Shapiro, 1970: Selikoff ct (I/.. 1972: Wagner, 1972) and includes intense fibroblastic activity in the lung, the formation of pleural plaques, and a singular relationship to pleural and peritoneal mesotheliomas. Persons with high exposure to airborne fiber who also smoke cigarettes had a greatly increased incidence of lung carcinoma (Selikoff et (il., 1964 and Hammond and Selikoff. 1973). Hazards of orally ingested asbestos fibers are less thoroughly understood, and the extent of uptake from the human intestinal tract is uncertain. Evidence that chrysotile and amphibole fibers penetrate the intestinal mucosa of the rat exists (Pontefract and Cunningham, 1973: Storeygard and Brown. 1977), and the oral intake of large amounts of chrysotile asbestos results in mucosal cytotoxicity in the same animal (Jacobs ct nl., 1977). However. actual penetration and systemic uptake is questioned by others (Smith, 1973: Gross clr cl/., 1974). and to date. long-term animal feeding experiments have failed to demonstrate tumor induction

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or other gross pathologic change. Fibers have been found in the urine of normal persons with oral amphibole exposure (Cook and Olson, 1979). as well as in stomach tumors and adjacent gastric mucosa in Japanese subjects with oral exposure to chrysotile (and presumed amphibole) (Henderson ef al., 1975). The incidence of gastrointestinal malignancies is higher in persons with inhalation exposure and probable oral exposure as well (Hammond et NI.. 1965). Because asbestos fibers are being identified with increasing frequency in potable water supplies, the extent of human intestinal uptake of these fibers and their potential consequences are of interest. We wish to report the identification of a particular amphibole fiber in the tissues of persons with a high oral intake of that fiber type. In 1973, this fiber, ferromagnesium silicate (Cummingtonite-Grunerite), was identified in the water of Lake Superior, which is used as the potable supply for Duluth, Minnesota, and adjacent communities (Cook et trl., 1974). Smaller amounts of other fibrous silicates also were found. Total counts of these amphibole fibers ranged from approximately 2 x 10” to 2 x 10” (average 2 x 10;) fibers/liter. The source was a taconite processing plant that discharged its tailings into the lake where prevailing currents carried the suspended microfibers toward the Duluth area. The average particle size in the water was less than approximately 1.5 x 0.2 pm. Data on the amount of tailings discharged since the plant began operations in 1955 indicated that the residents of Duluth had been exposed to these levels of fibers orally since the early 1960s. In the remainder of this report the term “amphibole fiber” refers only to the above-mentioned ferromagnesium silicate. METHODS

Cases for study were selected in a completely blind, random fashion from a pool of 239 cases involving residents of Duluth, Minneapolis, and St. Paul, Minnesota, and Houston, Texas, on whom autopsy was done in 1973 and 1974. The code was not broken until completion of analyses to avoid possible bias in evaluating the electron microscope grid preparations. Initially, it was hoped that non-Duluth residents coming to autopsy in Duluth could be used for control purposes. This was not done, however, because it was impossible to rule out antemortem fiber exposure during hospitalization in or during prior visits to Duluth. Therefore, control cases were selected from the Minneapolis-Saint Paul or the Houston areas, regions with a very low level of naturally occurring asbestos fibers in their water supplies. Thirty-three of the selected cases were of Duluth residents, but one case was later excluded because of residence in a home using well water instead of city water. Twenty-three (72%) of the Duluth group were females, and the average age was 72 years (52 to 89). The high average age reflected the large relative number of elderly persons in the general and hospitalized population of Duluth. None had industrial asbestos exposure. Twenty-one of the selected cases were from the control cities: 12 (62%‘) were females and the average age was 56 years (17 to 77), reflecting the younger average population age in these larger metropolitan areas, as well as the inclusion of a higher number of coroner’s cases in the autopsy material. All tissue samples, whether Duluth or control, were taken at autopsy according

ASBESTOS

FIBERS

IN

HUMAN

TISSUE

87

to a predetermined protocol and were preserved immediately in 109 formaldehyde. For liver and lung samples, tissue weighing approximately 0.1 g was removed from the center of the available specimen in order to minimize possible contamination from exterior surfaces which might have occurred at autopsy. The specimens of jejunum were washed thoroughly in filtered water before the removal of an approximately 0. l-g full-thickness portion of the wall. Early in the study, tissue specimens were ashed at 500°C for 18 hrs and the ash was resuspended in a measured volume of filtered water (generally 1 or 5 ml) using ultrasonic agitation. From these suspensions, lo-p1 aliquots were evaporated to dryness on each of several carbon-coated. Formvar-covered, 200-mesh electron microscope grids. Because excessive ash aggregation occurred in some of the initial preparations, thus interfering with small fiber identification. the preparation technique was modified to include predigestion of the tissue in 105 KOH and ultracentrifugation at 1.3 x 10” x 2 for 2 hr after the pH was adjusted to 7.4 ? 0.3. Ashing of the pellitized material and the preparation and examination of the electron microscope grids was not changed. In every case, the volume of solution evaporated on each grid was an accurately measured 10 ~1. Thus, the risk of contamination was held constant. From 1 to ten (average five) grid squares were examined at 10,000 to 20,000~ using a JEM IOOC microscope equipped with an energy dispersive X-ray analysis system. Three criteria were used to identify specific fibers. Only particles with approximately parallel sides, blunt ends, and aspect ratios 2 3: 1 were considered fibers. Only fibers with electron-diffraction patterns typical for chrysotile or amphibole asbestos were classified as asbestos fibers. and of this group, only those with Mg:Fe:Si ratios typical for the Cummingtonite-Grunerite species present in Duluth tap water were classified as ferromagnesium silicates and denoted as “amphibole fibers” herein. There was no possibility of confusing amphibole fibers with larger chrysotile fibers because the presence of a significant iron peak on dispersive X-ray analysis was required before a fiber was classified as amphibole. Fibers with the characteristic structure and with electron-diffraction patterns typical of chrysotile were included in that category without dispersive X-ray analysis if they were very small. Magnesium is known to leach from such fibers in the tissue milieu (Langer rt (II., 1971, 1973), and it was often impossible to demonstrate a significant magnesium peak on X-ray analysis on fibers less than 0.3 x 0.05 pm. All solutions used were first filtered through Nucleopore filters with a pore size of 0.1 pm. Laboratory contamination was monitored by preparing “blank” grids throughout the study. These were processed with the same solutions and techniques as were used for tissue ashing, but no tissue was actually included. In all. 65 “blank” grids were prepared and examined throughout the experiment. The ability of the analytic procedure to identify fibers in tissue was established by perfusing preparations from Duluth water of known fiber content through the portal vein of normal rat livers. After fixation, 0. I-g portions were analyzed periodically throughout the study. Recovery rates averaged 35 _t 15% (range 13 to 70%) for tissue, with fiber contents ranging from 1 x 10; to 5 x 10” fibers/g. Because tissue from exposed and control cases were obtained at different geographic sites, a check was made to determine if air-borne or water-borne fibers

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from the pathology facilities of a Duluth hospital would pose a significant problem of contamination. Normal fresh rat tissues were washed in 100 times their volume of water containing 2 x lox amphibole fibers/liter and were allowed to soak in such a solution for an hour before fixation. With the processing techniques employed, no fibers were subsequently identified in specimens from four lungs. three jejunums, and three livers. STATISTICAL

CONSIDERATIONS

Two estimates were used in evaluating tration of fibers was estimated as:

the findings of this study. The concen-

c, = No. of fibers identified

ml of solution examined

The volume of solution examined form of C, used therefore was:

was lo-” ml/grid

F

13 or 1O-S ml/grid

square. The

C = No. of fibers identified x IO” F x IO” = ___ (per ml). .\ ’ No. of grid squares examined When “blank” grids were examined, C, was the measure of laboratory contamination. When tissue grids were used, C, was a measure of both contamination and fibers from the tissue. The second estimate was the concentration of fibers per gram of wet tissue: C,=FxVxSxlO;’ 2

s x 1’ x M

(per g of tissue)

in which F. s, and 1’ are as given above, and V = volume of solution used to dissolve ashed specimen (ml), M = weight of tissue ashed (g), and S = total area of a single grid = lo” times the area of a single square. The distribution of C, for the 65 blank grids examined did not follow a Poisson distribution because of too many large values (see results below). In 24 of the tissue specimens examined, chrysotile fibers appeared occasionally as clumps of more than 50 fibers, again making the use of the Poisson distribution theory impossible. With the analytic procedure used, identification of a single fiber per electron microscope grid square corresponded to a tissue concentration of between 2 x 10’ and 1 x 10’; fibers/g of tissue, depending on whether the tissue ash was resuspended in 1 or in 5 ml of water. It is apparent that such individual measures of concentration are very crude and that averages must be used for meaningful comparisons. RESULTS

Basic observations and average rates (C, and C,) are shown in Table 1. Luhoratoyy c.otztnminLltiorz. Three-hundred grid squares on 65 “blank” grids were examined. There were 401 chrysotile fibers (1.3igrid square) but only one

Liver Live1

Control Duluth

‘I Column I’ Column ” Column “Column

L- (4)).

300

-

I (4)).

94 210

95 21s

84 107

Grid squares examined

(2)

20 32

21 33

20 32

(6) z (5)/(2). (7) = ((5) _. (3))/((2) (9) = (g)/(2). (IO) = ((8) * (3))/((2)

Jejunum Jejunum

Control Duluth

Blanks

Lung Lung

Type

Control Duluth

Source

Tissue

(1) No. of tissue specimens

Observations

76 52

97 44

100 I20

1 76 1

-

0 50

I 19

(3 Fibers identiiied

--.- 17 3.21

3.11 3.20

2.32 3.39

w

(4) Total tissue

0.003

0.01 0.36

0 0.23

0.01 0.46

-

0.36 5.86

0 .T.-‘0

0.51 16.21

401

431 420

310 223

1112 3457

(8) Fibers identitied

1.34

4.59 2.00

3.26 1.04

13.24 32.3 I

(9)” c, x 10-i (10~” x concentrationiml of solution examined)

(7)” cz x IO-’ (lo-” X concentration/g tissue)

(‘3”

(3) Total solution used for ash resuspension (ml) c, x 10 i (10 i x concentration/ml of solution examined)

Chrysotile

(concentrations)

Amphibole

observed

made

Asbestos

153.5 32.4

150.0 14.3

570.6 1143.7

cloy x 10-3 (10 i x concentrationig tissue) c,

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amphibole (O.O03/grid square) identified. Early in the study, 10 consecutive blank grids involving 52 grid squares seemed to be unusually contaminated because 158 chrysotile fibers (3.0/grid square) were found; all of the rest were approximately 1 fiber per square. In this report. all 65 “blank” grids have been included in spite of this apparent anomaly. The tissues processed at the same time did not appear to have unusually high concentrations of chrysotile. Ti.s.srrc~fiher c~tzfrnr. Of the 96 tissue specimens from Duluth residents, 60 had 1 amphibole fiber or more present compared with only 2 of the 61 tissue specimens from control cases (P < 0.001. Table 2). The lung appeared to have the highest burden (66% of specimens having at least 1 fiber). followed by the liver (63%) and the jejunum (59%‘). However, these percentages do not differ significantly among themselves. The average concentration of amphibole in Duluth cases ranged from approximately 3 x IO” fibers/g (jejunum) to 16 x IO” (lung). %th to li70th the concentrations of chrysotile identified in the same specimens. The amphibole concentration in controls was approximately that of the blanks and can be considered to be due to contamination alone. Chrysotile fibers were found in the lung tissue from all control cases and in 30 of 32 Duluth cases (Table 2). Levels of chrysotile in the lung were significantly TABLE

2

DISTRIBUTION OF CONCENTRATION IO- ’ g TISSLIE BY TYPE ANII

Lung Concentration of fibers

Duluth

__~~ Control

OF ASBESTOS FIBERS SOUK~L or TISSUE

PER

Liver

Jejunum Duluth

Control

Duluth

Control

Amphibole (per gram) 0 0 to 19.9 20 to 49.9 50 to 99.9 loo to 199.9

II 8 IO 3

19 1 0 0

13 18 0 I

21 0 0 0

I2 18 0 1 1

19 1 0 0 0

Total ( ; ‘,. 0

32 66

20 5

32 59,

21 0

32 63 ’

20 5

Chrybotile ( * IO") 0 0 to 19.9 20 to 49.9 50 to 99.9 100 to 199.9 200 to 399.9 3400 (clumping)

2 4 3 2 4 3 14

0 1 1 4 2 7 5

4 22 4 0 1 0 I

I 3 5 2 5 3 2

4 21 2 3 1 0 !

0 5 4 4 6 0 2

Total

32

20

32...‘

21

32-A.

21

* Significantly ** Significantly

different different

from from

control control

(P < 0.001) (P < 0.01).

ASBESTOS

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91

greater than contamination levels. Lung specimens in seven Duluth cases and one control case had large numbers (more than 100) of chrysotile fibers occurring in bundles and clumps, making an accurate count of total fibers very difficult. These cases accounted for the large apparent excess of chrysotile in the Duluth specimens shown in Table 1 (5.7 x lO’/g controls, 1.14 x lO”/g Duluth). Various nonparametric statistical tests showed that this difference was not significant. In contrast to the lung specimens, jejunum and liver samples in Duluth cases had significantly less chrysotile than did the control cases (P < 0.011. In fact, these chrysotile levels in Duluth cases did not differ significantly from the “blank” grids: hence, these chrysotile fibers could reasonably have been due to laboratory contamination alone. The average concentration of chrysotile in control jejunum and liver specimens was approximately I .5 x IO”/g, li40th of the lung burden. DISCUSSION

This study demonstrates that about 60c’,/r of a group of persons with long-term oral intake of a particular amphibole fiber have this same fiber type present in lung, liver, and intestinal wall in levels greater than 2 x 10’ fibers/g. Presumably. this tissue burden resulted from transmucosal intestinal uptake of the fibers. Several air sampling studies were done in the Duluth area starting in 1973, and in no instance were appreciable numbers of amphibole fibers found in the air in tandomly selected homes or in out-of-doors samples t Environmental Research Laboratory et al., unpublished data). Although in Duluth cases, the amphibole fiber levels in lung were a little higher than the levels in either liver or jejunum, the differences were not significant statistically and could have resulted from sampling variation, as well as from biologically determined differences in the distribution of absorbed fibers. We believe that no inconsistency exists between the data reported herein and the results of a recent study by Auerbach c~fI//. (1977), which showed that the number of ferruginous bodies in lung tissue sections of Duluth cases was essentially equal to that for New York City residents. Because both chrysotile and amphibole fibers can result in the formation of ferruginous bodies detectable by light microscopy and because the chrysotile content of Duluth lung specimens was comparable to that found in two other large metropolitan areas, the relatively small additional number of specific amphibole fibers in Duluth specimens could easily have been obscured in any analytic technique that could not identify specific fiber mineralogy. From the present data, it is impossible to predict whether the amphibole levels in tissue of Duluth residents are steadily increasing as the duration of oral exposure lengthened, or whether they represent some type of steady-state. balancing absorption and urinary fiber excretion which occur under some circumstances (Cook and Olson, 1979). The long latent period between the initial exposure to asbestos and the appearance of asbestos-related disease, together with the known cases in which brief exposures have resulted in asbestos-related disease years later, argue against the elimination of hazardous tissue levels through any mechanism of effective excretion. Recent studies have shown no excess of gastrointestinal malignancies in Duluth residents through 1974, compared with residents of Minneapolis-Saint Paul, Minnesota (Levy et al.. 1976). However, the number

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of years that have elapsed since the start of oral amphibole exposure for the Duluth residents is still too few to allow any conclusions about ultimate risk. In 1976, a primary water filtration plant began operation in Duluth and has proved to be 997~ effective in removing the amphibole fibers from the potable water supply. It will be important to determine if future tissue specimens show a reduction in fiber burden. In our opinion, the fact that the control cases selected through blind sampling in this study involved younger subjects than did the Duluth cases does not invalidate the significance of the results obtained. No age-related trend in amphibole or chrysotile concentrations for either Duluth or control specimens was noted. Agerelated effects have not been reported for other forms of particulate from the intestinal tract. Also, the time of exposure in Duluth to the specific amphibole fiber in question was no longer than 20 years and probably less than 1.5 years for the high concentrations, a span much shorter than the average adult life span of the control subjects. To make certain. however, that the age difference between the experimental groups did not alter the significance of the findings, the data were reanalyzed using only those control and Duluth cases matched for age. Twentytwo Duluth subjects and 14 control subjects in the age range from 50 through 79 years were included. One amphibole fiber was found in a single tissue from each of two control subjects (ages 61 and 66 years), while one or more amphibole fibers were found in 44 of 66 tissue specimens from the 22 Duluth subjects, a difference that was highly significant statistically V’ < 0.001). No explanation presently exists for the low level of chrysotile found in liver and jejunum specimens from the Duluth subjects. To the best of our knowledge, chrysotile levels were low in the water supplies of all areas used in the study. SUMMARY

1. A specific type of amphibole fiber was found in significant numbers in the lung, liver, and jejunum in residents of Duluth, Minnesota, who had oral exposure to high levels of these mineral fibers for up to 15 years. 2. Although the differences in concentrations of this fiber type among the tissues studied was not significant statistically, the lung appeared to have the highest burden, followed by the liver and jejunum. 3. Chrysotile fibers were found in relatively high concentration in lung specimens from both Duluth and control subjects. The chrysotile content in liver and jejunum samples taken from Duluth subjects did not differ significantly from background levels resulting from laboratory contamination, while the liver and jejunum specimens from control subjects had a small but significantly higher chrysotile content. ACKNOWLEDGMENTS The assistance and ronmental Protection gratefully recognized, the first phases of this

cooperation of Mr. James Tucker. Environmental Research Laboratory, EnviAgency, in making electron microscopy facilities available for this study is Mrs. Eileen Gannon and Mr. Arnie Pozos gave valuable technical assistance in experiment.

REFERENCES Auerbach. 0.. Hammond, E. C., Selikoff, Asbestos bodies in lung parenchyma Et~\~irot~. Rrs. 14, 286-304.

I. J.. Parks, in relation

V. R.. Kaslow. H. D.. and Gartinkel. L. (1977). to ingestion and inhalation of mineral fibers.

ASBESTOS Cook.

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P. M.. Glass. G. E.. and Tucker, J. H. (1974). Asbestiform amphibole minerals: Detection and measurement of high concentrations in municipal water supplies. .S<,irnc,e 185. 853-855. Cook. P. M. and Olson, G. F. (1979). Ingested mineral fibers: Elimination in human urine. .S(~ic,~)c~~, 204, 195- 198. Cunningham, H. M.. and Pontefract. R. (1971). Asbestos tibres in beverages and drinking water. Nnfrrre (f.ondon) 232, 332333. Cunningham. H. M.. and Pontefract. R. D. ( 1973). Asbestos fibers in beverages. drinking water. and tissues: Their passage through the intestinal wall and movement through the body. .I. Ass. Qffic.. AM/. Clrcm. 56, 9766981. Gross. P., Harley. R. A.. Swinbume. L. M.. Davis. J. M. G., and Greene. W. B. (1974). Ingested mineral fibers: Do they penetrate tissue or cause cancer? Arch. Environ. Health 29, 341-347. Hammond, E. C.. and Selikoff. I. J. (1973). Relation of cigarette smoking to risk of death of asbestosassociated disease among insulation workers in the United States. III “Biological Effects of Asbestos” (P. Bogovski, J. C. Gilson, V. Timbrell. and J. C. Wagner. Eds.). pp. 312-317. International Agency for Research on Cancer. Lyon. Hammond. E. C., Selikoff. I. J.. and Churg. J. (1965). Neoplasia among insulation workers in the United States vvith special reference to intra-abdominal neoplasia. Atrrr. N. I’. Actrcl. SC,;. 132, 5199525. Henderson, W. J., Evans. D. M. D.. Davies. J. D.. and Griffiths. K. (1975). Analysis of particles in stomach tumours from Japanese males. Ert),iro,r. Rcr. 9. 240-249. Jacobs. R.. Dodgson, K. S., and Richards. R. J. (1977). A preliminary study of biochemical changes in the rat small intestine following long-term ingestion of chrysotile asbestos. Brir. J. E.\p. furho/. 58. 541-548. Langer. A. M., Rubin. I. B.. Selikoff, I. J.. and Pooley. F. D. (1972). Chemical characterization of uncoated asbestos fibers from the lungs of asbestos workers by electron microprobe analysis. .I. Hisr~~c~/fc~m. C\‘fw/lc’m 20. 735 -740. Langer, A. M.. Selikoff. I. J., and Sastre. A. (1971). Chrysotile asbestos in the lungs of persons in New York City. AK/I. Eu~.ir~~tf. N~trlrh 22. 348-361. Levy. B. S.. Sigurdson. E.. Mandel, J.. Laudon. E.. and Pearson. J. (1976). Investigating possible effects of asbestos in city water: Surveillance of gastrointestinal cancer incidence in Duluth, Minnesota. Arrw~. J. Epi&,rnid. 103. 362-368. Nicholson. W. J. (1974). Analysis of amphibole asbestiform tibers in municipal water supplies. E11r.i. rotz. Hvtrlth Pcrrpw~. 9. 165- 172. Nicholson. W. J., and Pundsack, F. L. (1973). Asbestos in the environment. /jr “Biological Effects of Asbestos” (P. Bogovski. J. C. Gilson. V. Timbre]]. and J. C. Wagner. Eds.), pp. l?6- 130. International Agency for Research on Cancer. Lyon. Pontefract. R. D.. and Cunningham. H. M. (1973). Penetration of asbestos through the digestive tract of rats. ,Ytrrrrrc, IL~I&I~IJ 243, 352-353. Selikoff. I. J.. Churg, J.. and Hammond, E. C. (1964). Asbestos exposure and neoplasia. J. Amvr. Med. A.\\. 188. 22-26. Selikoff. 1. J.. Hammond. E. C.. and Churg. J. (1972). Carcinogenicity of amosite asbestos, ~lc./r. E~~~irrm. H~,tr/th 25. 183 - 186. Shapiro, H. A. (1970). “Pneumoconiosis.” Oxford Univ. Press. London. Smith. W. E. (1973). Asbestos. talc and nitrites in relation to gastric cancer, ,4rn(lr. f,)c/. H~,s. A.\.\. J. 34. 2277228. Storeygard. A. R.. and Brown. A. L.. Jr. (1977). Penetration ofthe small intestinal mucosa by asbestos fibers. Mt1!0 C/i,). &(I(,. 52, 8099812. Wagner. J. C. (1972). Current opinions on the asbestos cancer problem. A,rrr. Oc.c,rr/). H?p, 15.61-65.