Assessment of reference values for elements in hair of urban normal subjects

MICROCHEMICAL

JOURNAL

Assessment

%CAROLI,*

46,

174-183

(1992)

of Reference Values for Elements of Urban Normal Subjects’

O.SENOFONTE,N.VIOLANTE, Istituto

Superiore Received

di Sanitci, January

Vi&

in Hair

L. FORNARELLLAND Regina

2, 1991; accepted

Elena

299, 00161 Rome,

February

A. POWAR Italy

19, 1992

Hair is quite attractive as a biological indicator because of the simplicity of sampling and storage. The analysis of trace and minor elements in this tissue has been used in a diversity of applications, including nutritional, environmental, medical, and occupational studies. Within this context the availability of reliable reference values plays a key role. Scalp hair samples taken from 225 urban subjects not older than 15 years were analyzed for 18 elements. The method chosen for the quantification of elements in hair has been ICP-AES in all cases because of its multielemental capability, wide dynamic range, adequate detection limits, and relative freedom from matrix interferences. The following mean values were obtained (in pg g-l): Al, 11.6; As, 0.10; Ca, 416; Cd, 0.20; Co, 0.86; Cr, 0.67; Cu, 22.4; Fe, 17.9; Mg, 25.9; Mn, 0.41; MO, 0.44; Ni, 0.97; P, 185; Pb, 8.10; Se, 0.84; Ti, 1.05; V, 1.44; and Zn 144. These data were analyzed statistically to ascertain possible significant differences according to sex and age. o I!W Academic PWS, IK.

INTRODUCTION The determination of trace elements in biological fluids and tissues not only is a means for evaluating actual exposure events, but also has the potential for revealing and reconstructing past episodes relevant to health even after their action has ceased. Hair, a highly stable material that can be painlessly collected and easily stored, exhibits the ability to play a role in this detective story. This kind of tissue lends itself to biomedical, epidemiological, and environmental studies for monitoring and assessing exposure to noxious pollutants (Z-3). In fact, there are at least two main advantages inherent in this type of analysis, which can be traced respectively to the fact that hair reflects total body intake of certain elements better than the more often employed blood and urine and to the accumulation of inorganic components over extended periods of time, which leads to analyte concentrations higher as a rule by one order of magnitude than those in biological fluids. Despite these advantages, hair analysis has not yet gained the popularity and general acceptance it deserves. Historically, the first discipline to benefit from hair analysis was forensic science in that it was deemed possible to reconstruct a given criminal episode by identifying the elements present in a few strands of hair. Far from being simply a literary artifact to highlight the perspicacity of the protagonist of a mystery novel, hair analysis has often played a major role in unraveling the plot, as was recently the case, e.g., with an episode of As poisoning (4). ’ Submitted in conjunction with the Fifth Italo-Hungarian Symposium Control and Assurance in Life Sciences, Pisa, Italy, September 9-13, * To whom correspondence should be addressed. 174 0026-265X/92 Copyright AI. A-L-^

$4.00

Q 1992 by Academic Press, Inc. ^.-_^__^A ..^. :^_ z_ ^_.. r------..-A

on Spectrochemistry: 1991.

Quality

REFERENCE

VALUES

FOR

ELEMENTS

IN

HAIR

175

Elements can find their way into hair through a multiplicity of sources, both endogenous and exogenous (5, 6). The endogenous sources are definitely more important than the exogenous sources for evaluating the health status of an individual with regard to physiological anomalies, nutritional imbalances, or uptake of environmental toxicants. From this point of view, substances transported by the bloodstream contribute dramatically to the buildup of the elemental burden, even though the hygroscopicity of hair greatly favors the incorporation of contaminants from external agents such as water, shampoos, and cosmetic preparations of any sort. As reported by Hopps (I), trace elements can enter the hair continuously during its growth, and thus variations in the element concentrations of circulating body fluids are recorded along its axis. Especially significant are the amounts of elements conveyed by sebaceous excretion as well as through eccrine sweat, which also acts as a leaching factor. Incorporation of elements into the keratin structure of hair takes place through binding to the sulfhydryl groups abundantly present in the follicular proteins. Detergents, such as soaps and shampoos, cold-waving lotions, hair bleachers, and dyes actually compete with the complexing ability of these reactive sites and thus lead to a significant depletion of elements from the bulk shaft. On the other hand, the presence of a substantial load of inorganic contaminants in the various environmental compartments inevitably imposes the conditions for their deposition onto, and eventually absorption through, the hair surface. This phenomenon adds to the exogenous accumulation caused by the already mentioned (and intentional) cosmetic treatments as well as by contact with desquamating epidermis. The type of hair sample selected is also a matter of debate, whether pubic, axillary, limb, or scalp. Each has its own analytical merits, but is also plagued by some disadvantages. Any sound decision in this sense relies on the elements to be analyzed and the suspected impact of exogenous agents. Although scalp hair is more prone to contamination through environmental conditions and cosmetic treatments, it is still preferable to hair from other body parts in that it is less affected by natural excretions. Multielemental analyses of hair are needed for the following equally important reasons. Because the number of elements with potential toxicity is high, this approach permits antagonistic and synergistic aspects to be taken into account (7, 8). “Normality” is a frustrating concept that lacks a satisfactory definition. What should be considered normal concentrations for certain elements in hair strongly depends on the assumed absence of perturbing factors such as local emissions and toxicants, diet imbalances, pathological conditions, and the like. Thus, it makes much more sense to speak generally of reference values (RVs) for the element concentrations, those that can be assigned to individuals whose environmental exposure and health risk on the whole do not affect the standards considered acceptable in terms of welfare and life expectancy. As for all other tissues and organs, a reliable use of elemental analysis in hair is unavoidably bound to the knowledge of such RVs and reference intervals (RIs) for the analytes of interest for well-characterized population groups. Several national and international organizations have undertaken programs to collect such data. The activities of the International Atomic Energy Agency (IAEA), which started

176

CAROL1

ET

AL.

in 1977 a special subprogram named Health-Related Environmental Research, are particularly noteworthy. This has also led to initiation of the project NuclearBased Methods for the Analysis of Pollutants in Human Hair (9). Another example of striking importance is the initiative taken a few years ago in the UK to assess the suitability of this kind of investigation for gaining information on the health status of the fetus in early pregnancy and relating it to the preconception situation (10). More recently, a campaign has been promoted by our research group to establish the RVs for a number of minor and trace elements in healthy subjects up to 15 years old in the urban area of Rome. Given the young age of the donors, it is thought that the data arrived at will better reflect the intrinsic concentration ranges, as these are less likely to be influenced by cosmetic treatments. This paper thus reports the results obtained so far for a group of 225 subjects. At the same time, differences between adult and elderly groups will be reliably detected when the study is extended to other age categories, as planned. The strategy and the data obtained so far in this investigation are described in detail in the following. MATERIALS

AND METHODS

(A) Sampling All facets of the collection campaign were carefully planned and are summarized below: (i) A a number of infant, primary, and secondary schools in central and semicentral parts of Rome were selected in order to set up a network of sampling sources that would reasonably reflect the average urban exposure not atfected by any specific emission of pollutants. (ii) Subjects selected were in no case older than 15 years and were determined with reasonable certainty to be free of major and minor pathologies. In this respect, the form shown in Table 1 was used during an interview to collect principal information on each youngster. (iii) The hair bundle was always cut from the same zone of the scalp for all subjects, namely, the occipital region, at a distance of approximately 1 cm from the scalp itself. Titanium nitride-coated scissors were employed throughout the campaign to minimize any possible release of contaminating elements during this crucial phase of the overall procedure. (iv) Hair (1 to 2 g) thus cut was immediately placed in polyethylene bags, which were then accurately sealed and labeled with a progressive number, the subject name, and the date. (v) All specimens were stored in a dry, cool, and ventilated environment until delivery to the laboratory and then kept in desiccators until analysis. The above steps are rigorous enough to fully guarantee the reliability and validity of the operational approach adopted, and at the same time do not impose an excessive load in terms of practicability. (B) Sample Treatment One of the more delicate phases of sample pretreatment

consists of the removal

REFERENCE

VALUES

FOR

ELEMENTS

IN

177

HAIR

of exogenous substances from the hair surface. Sample preparation is very critical, as washing procedures may well leach a number of elements at different rates. Dirt and dust, on the other hand, are relatively easy to remove. No standard procedure for hair washing and pretreatment can be prescribed in general: it can only be recommended that it be as simple as possible and that the ultimate goal not be the complete removal of environmental contaminants for the reasons mentioned above. Further, the selection of a strategy also depends on the analytical technique to be employed, as this determines the final form in which the samples must be presented. A variety of chemical and physical treatments have been devised so far to circumvent the difficulties inherent in hair washing, as abun-

TABLE

Information Subject

Form

Employed

1

in the Sampling

Campaign

No.-

Family

Christian

Name:

Name:

Address: Province:

City: Sex:

(male)-

Weight:Health

Comments

(female)-

Age:-

Height:-

(kg)-(h) conditions

(brief

food

on

(months)

(years)--.(m);

-(cm)

description):

habits

and

life

customs

in

general

(brief

description):

Specific application,

remarks

(e.g.,

and the

type like):

of

shampoo

normally

used,

frequency

of

178

CAROL1

ET AL.

dantly described in the relevant literature [see, e.g., Refs. (II, 12)]. Among these, mention should certainly be made of the rather popular use of EDTA as a complexing agent to detach loosely adhering metals. This treatment, however, should be applied with the greatest care; otherwise, it could hinder obtaining information from the inner layers of the hair shaft. Under the constraints of two contrasting needs-cleaning of the samples without altering their real content of elements-a mild, yet efficient procedure has been developed. This involves a thorough washing of about 0.5 g of hair, cut in pieces no longer than 1 cm, with a mixture of ethyl ether and acetone (3 + 1, v/v) under continuous stirring for 10 min; drying at 85°C for 1 h; treatment with a diluted (5%) aqueous solution of EDTA for 1 h; repeated rinsing with double-distilled water; and finally drying at 85°C for 12 h in an oven to determine the sample dry weight just before the subsequent step is started. Hair digestion has also been carried out using a multiplicity of methods of both wet and dry ashing type. Given the high number of samples to be treated (on the order of TABLE MW

Sample

Digestion

Treatment

Sequence

CEM MDS

MW Oven

weight:

containers:

steps:

2

Digestion

81D

approximately

1 g

white

Teflon,

capable

of

overnight purity, an

pressure-tight sustaining

about

predigestion

with

concentrated MW power

MW power

of of

addition 30 1

h

min

quantitative tubes

Capacity:

12 rotating

at

W;

30

min

stage

at

W;

vessels

at at

carousel

q in

cooling;

and

an

1 q L HF;

MW power

an into

up

placed

10

H202

stage

dilution

high-

stage

transfer and

mL of min

mL

stage

10

stm

30

240

1

6-10

HNO3; 180

ca.

of

further and

ca.

vessels

300 360

MW polypropylene

to

on

20

a

mL

continuously

W W;

REFERENCE

VALUES

FOR

ELEMENTS

IN

179

HAIR

TABLE 3 Instrumental (A) and Analytical Conditions (B) for ICP-AES A

ISA Instruments Jobin-Yvon 32 t 38 VHR Spectrometer,consisting of a polychromator (HR 1000 M, focal length O.&a, Pasch n-Runge mounting, holographic concave grating of 3 00 grooves mm-f , linear P dispersion in the first order 0.55 nm mm- , spectral range 170 410 nm, with 9 channels) and a monochromator (HR 1000 M, focal length 1 m, Czerny-Turner mounting, holographic plane grating of 3600 grooves mm-', linear dispersion in the first order 0.27 nm mm-l, theoretical resolution 504,000, spectral range 170 - 470 nm). HF Generator DURR - JY 3848, with a frequency of 56 MHz and a nominal output of 2.2 kW INSA demountable torch Hydride generator system PS Analytical with continuous flow Computer IBM, Software JY 3.5. B

q

Torch argon flows, 16 L min-l(outer) and 0.6 L in-' (intermediate) Nebulizer flow, 0.6 L rain-' Entrance slits, 50 pm (polychromator) and 40 pm (monochromator) Exit slits, 50 pm (polychromator) and 40 pm (monochromator) Spectral lines (in nm), Al 396.2, As 193.7, Ca 317.9, Cd 226.5, Co 238.9, Cr 267.7, Cu 324.8, Fe 259.9, Mg 279.6, Mn 257.6, MO 292.3, Ni 231.6, P 214.9, Pb 220.4, Se 196.0, Ti 336.1, V 292.4, Zn 213.9

TABLE 4 Element Content of Hair for All Subjects Concentration Element

Mean

Median

Al As Ca Cd co Cr cu Fe Mg Mn

11.59 0.099 416.2 0.205 0.86 0.67 22.38 17.87 25.87 0.41 0.44 0.70 185.1 8.10 0.84 1.05 1.44 144.2

10.6 0.074 242.0 0.140 0.20 0.40 10.0 13.61 21.53 0.28 0.21 0.35 185.7 7.20 0.57 0.75 1.06 143.2

MO

Ni P Pb Se Ti V Zn

(pg/g

5th-95th 2.5 0.01 167.9 0.041 0.035 0.085 7.17 5.79 8.27 0.043 0.043 0.05 88.9 1.13 0.26 0.13 0.04 53.7

dry

Percentiles -

20.05 0.27 975.8 0.52 3.06 2.54 89.40 37.47 59.22 1.11 1.42 2.92 327.6 21.1 1.52 2.64 3.69 230.2

weight) Range 0.21 0.002 63.6 0.028 0.024 0.03 1.69 3.54 0.32 0.03 0.025 0.027 18.2 0.53 0.049 0.041 0.03 23.8

spanned -

22.6 0.85 3102 1.72 16.2 11.3 279.6 215.6 137.5 7.59 6.90 10.02 511.7 31.1 9.79 13.05 22.91 476.8

180

CAROL1 ET AL. TABLE 5 Element Content of Hair According to Sex Concentration Element

AS

Ca Cd co Cr CU Fe hl Mn MO

Ni P Pb Se Ti V 2n

Median

Mean Males

Al

(pg/g dry weight)

Females

11.53 0.103 351.1 0.169 0.932 0.779 24.46 18.39 22.11 0.416 0.411 0.701 195.0 7.750 0.820 1.040 1.330 141.3

Males

11.68 0.092 507.8 0.258 0.731 0.545 19.48 17.12 31.07 0.399 0.479 0.778 171.3 8.578 0.874 1.061 1.638 147.3

a

Females

10.60 0.050 300.9 0.128 0.190 0.320 9.664 13.61 19.20 0.298 0.217 0.400 195.8 6.325 0.567 0.692 1.084 139.4

9.684 0.070 444.2 0.140 0.20 0.450 10.65 13.40 25.53 0.250 0.210 0.410 156.5 8.140 0.557 0.870 0.810 144.4

> > < < > > > > < > < > > < > > > >

a P value as deduced by Student’s t test.

TABLE 6 Element Content of Hair According to Age Concentration

Element Al As Ca Cd co Cr CU Fe a Mn MO

Ni P Pb Se Ti

V Zn

(p&g

Mean 3-6 years 15.65 0.110 323.3 0.152 0.472 0.880 24.22 13.34 20.58 0.360 0.548 1.529 147.8 9.391 0.865 1.179 0.255 101.1

dry

weight1

Median 9-13 10.16 0.096 447.9 0.230 0.954 0.559 21.74 19.48 27.72 0.426 0.401 1.349 198.1 7.646 0.838 1.005 1.685 159.1

years

a P value as deduced by Student’s t test.

3-6

a

years 14.60

9-13 years 7.900

0.080

0.090

287 .O 0.090 0.200 0.390 9.980 13.70 3.001 0.039 0.180 0.270 139.5 9.230 0.600 0.984 0.130 90.80

374.6 0.128 0.052 0.427 10.50 12.80 13.10 0.270 0.180 0.320 144.2 7.800 0.450 0.920 0.920 144.5

< > > > c < > > > > > > > > ) > < <

0.001 0.1 0.1 0.1 0.001 0.05 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.05 0.05

0.1 0.1 0.01 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Al As Ca Cd CO ClCU Fe Mg Mn MO Ni P Pb Se Ti v Zn

Element

0.21 0.002 63.60 0.028 0.024 0.030 1.69 3.54 0.32 0.03 0.025 0.027 18.20 0.530 0.049 0.041 0.030 23.8

(this

Italy

- 22.60 - 0.851 - 3102 - 1.72 - 16.2 - 11.3 - 279.6 - 215.6 - 137.5 - 7.59 - 6.90 - 10.02 - 511.7 - 31.10 - 9.79 - 13.0 - 22.91 - 476.8

study)

Concentration

-

-

1.79 0.10 150 0.112 0.012 0.026 4.57 5.17 30.37 0.208 0.027 0.44

0.340 1.11 0.011 141.9

(Ref.11)

England

2.83 8.93 0.080 259.6

9.43 2.41 1620 0.994 0.198 1.88 19.41 38.68 81.65 3.98 0.169 7.10 -

- 220

0.40 110 2.0 1.0

120

1.3 190 4.0 1.4

2220 0.43 0.23 0.41 18 15 160 0.36

1 0.10 0.20 0.20 6.5 4.0 0.06 0.06

14

1.0 -

0.60

(Ref.12)

USA

Concentration

0.564 - 357

0.005108

- 3.72

0.03

- 87.54

- 245

4

0.24

- 93.1

0.7

17

-

1

(Ref.131

Canada

(pg/g

dry

0.16 72

0.6 1 190 0.05 0.13 0.20 6.0 5.5 14 0.06 0.20 0.17 115 1.4 1.0

(Ref.14)

Japan

weight)

36 2.7 3700 0.57 0.49 0.77 69.1 87.4 567 4.51 0.59 3.0 250 18.0 4.9 - 0.88 - 327

0.233 1.42 0.019 157.6

1.05 8.39 - 0.080 - 293.2

-

- 10.8 - 1.05 - 1380 - 1.51 - 0.098 - 1.92 - 8.12 - 52.80 - 149.3 - 1.68 - 0.212 - 4.52

15)

Zealand

6.16 0.279 250 0.361 0.041 0.560 3.42 18.45 73.45 0.573 0.102 1.62

(Ref.

New

TABLE 7 Ranges for Elements in the Hair of Healthy Individuals from Different Countries

0.418 2.15 0.012 143.8

2.69 0.037 170 0.561 0.032 0.205 7.21 12.94 25.32 0.201 0.010 0.55

(Ref.

Bulgaria

-

2.45 5.84 0.360 284.4

- 21.3 - 0.625 - 1900 - 2.71 - 0.166 - 1.02 - 19.4 - 96.4 - 128.9 - 4.30 - 0.066 - 3.59

16)

182

CAROL1

ET

AL.

several hundreds) preference in this study has been given to a relatively new digestion approach based on irradiation in a microwave (MW) oven at 2.45 GHz, which considerably reduces the ashing time, as a rule by one order of magnitude. The system, supplied by the CEM Corp. (U.S.A.), allows programmable cycles of MW irradiation to be carried out, minimizing both duration and risk of contamination. The details of this procedure are summarized in Table 2. (C) Analytical

Determinations

The method of choice for quantitation of elements in hair adopted in this study has been in most cases inductively coupled plasma atomic emission spectrometry (ICP-AES) because of its suitability for this kind of investigation (multielemental capability, wide dynamic range, adequate detection power, and relative freedom from matrix interferences, among others). Occasionally atomic absorption spectrometry (ASS) measurements were carried out to check the reliability of data for certain elements particularly prone to spectral interferences with ICP-AES, such as Cr and Ni. The working parameters for this technique are summarized in Table 3. The entire analytical procedure was tested for both precision and accuracy of measurements in order to assess the degree of reliability of the data generated. The level of accuracy was continuously monitored by adding to each series of unknowns one of the two reference materials available, namely, the NIES (National Institute of Environmental Sciences, Tokyo, Japan) No. 5, certified for As, Co, Cd, Cu, Fe, Mg, Mn, Pb, Sb, Se, and Zn, and the BCR (Community Bureau of Reference, Bruxelles, Belgium) No. 397. The other basic requirement, i.e., precision, was ascertained by replicating the determinations an adequate number of times (not less than 10). Although both of the parameters are of fundamental importance, it should be stressed here that the very nature of the study makes accuracy all the more crucial in that it affects the confidence in the obtained reference values. Since these are the starting point for any further progress in the use of hair analysis for diagnostic purposes, it is immediately understandable that accuracy is of paramount importance. On the other hand, given the high number of subjects considered (225), individual variability makes achieving the highest possible precision unessential because of the much wider range of values expected for each element in the population examined. RESULTS

AND DISCUSSION

The data obtained so far provide a preliminary and sufficiently reliable indication of the concentration ranges to be established for the elements under test. An overall view of the main outcomes of this pilot study is set forth in Table 4, where the data reported refer to the entire test population. The uncertainty of each average value is in some cases even larger than the mean itself, thus implying negative figures for the lower extreme of the resulting interval. This is obviously a purely mathematical consequence of the statistical treatment applied to the experimental measurements and has no physical meaning because concentration cannot be negative. It should be kept in mind, therefore, that the calculated standard deviations simply reflect the distribution of the elemental contents for different individuals, i.e., the biological variability, rather than the precision of a

REFERENCE

VALUES

FOR ELEMENTS

IN HAIR

183

set of measurements repeatedly carried out on the same quantity. The real range of concentrations encountered for each element is shown in the last column of Table 4. On the other hand, the data listed in Table 5 seem generally to support the view that there are no striking differences for most elements between males and females. In a few cases, however, a borderline situation is apparent; with Ca, Cd, Mg, and MO, sex-specific variations begin to be appreciable. Definitely more interesting information can be obtained by comparing subgroups of subjects following the criterion of age in combination with that sex. Further, if the concentration values are split on the basis of age, as reported in Table 6, additional interesting remarks are possible. In fact, it is apparent that significant differences exist for most elements, with the exception of As, Cu, Ni, Se, and Ti. This should lend support to the hypothesis that the accumulation mechanisms do vary in an unforeseeable fashion depending on at least three paremeters, sex, age, and nature of the element. Although this is certainly acceptable in principle, the information obtained so far is too scanty to corroborate the hypothesis, and many more than the present 225 subjects would be necessary for that purpose. Finally, the values obtained in this study are compared with those available in the international literature, as shown in Table 7. Because there is definitely no abundance of the latter, the data reported therein are not homogeneous in terms of age, sampling procedure, and living conditions. Nonetheless, the comparison seems to point to surprisingly overlapping ranges except in a few instances, thus substantiating the uniformity of the actual accumulation process in an urban environment. To clarify whether this assumption is valid or must be modified to account for more complex and less understandable aspects is the aim of future phases of this investigation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Hopps H. C. Sci. Tot. Environ., 1977, 7, 71-89. Eads, E. A.; Lambdin, C. E. Environ. Res., 1973, 6, 247-252. Reusbew, B. D. Med. Sci. Lnw., 1976, 16, 37-39. Kohlwr-Schmidt, H.; Bohn, G. Determination of arsenic poisoning from traces in the hair after protracted time. In Topics in Forensic and Analytical Toxicology (R. A. A. Maes, Ed), pp. 39-44. Elsevier, Amsterdam, 1984. Moon, J.; Davison, A. J.; Smith, T. J.; Fadl, S. Sci. Tot. Environ., 1988, 72, 87-112. Evans, G. J.; Jervis, R. E. .I. Radioanal. Nucl. Chem. Art., 1987, 110, 613-625. Manson, P.; Zlotkin, S. Can. Med. Assoc. J., 1985, 133, 186-188. Laker, M. Lancer, 1982, 2, 260-262. Ryabukhin, Yu. S. International Coordinated Program on Activation Analysis of Trace Element Pollutants in Human Hair. In Hair, Truce Elements and Human Illness (A. C. Brown and R. G. Crounse, Eds). Praeger Publishers, New York, 1980. Barlow, P. J.; Sidani, S. A.; Lyons;, N. Sci. Tot. Environ. 1985, 42, 121-131. Ward, N. I. Personal communication. Data supplied by Mineral Lab, U.S.A. Ryan, D. E.; Holzbecher, J.; Stuart, D. C. Clin. Chem. 1978, 24, 19962000. Kamakura, M. Jap. J. Hyg., 1983, 32, 823-838. Ward, N. I. Conf. New Zealand Trace Elem. Group, Massey University, Palmerston North, New Zealand, Aug. 7-8, 1986. Ward, N. I.; Spyrou, N. M.; Damyanova, A. A. J. Radioanal. Nucl. Chem. 1987, 116, 125-135.