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You are on page 1of 0

LALMOHAN BHAR

Indian Agricultural Statistics Research Institute,

Library Avenue, New Delhi-110 012

lmb@iasri.res.in

1. Introduction

One type of assay which has been found valuable in many different fields, but especially

in toxically studies, is that dependent upon the quantal, or all-or-nothing, response.

Though quantitative measurement of a response is always to be preferred when available,

there are certain responses which permit of no graduation and which can only be

expressed as occurring or not-occurring. The most obvious example of this kind of

response is death; although workers with insects have often found difficulty in deciding

precisely when an insect is dead, in many investigations the only practical interest lies in

whether or not it has a test insect is dead, or perhaps in whether or not it has reached a

degree of inactivity such as is thought certain to be followed by early by early death. In

fungicidal investigations, failure of a spore to germinate is a quantal response of similar

importance. In studies of drug potency, the response may be the cure of some particular

morbid condition, no possibility of partial cure being under consideration. This lecture is

concerned with the statistical techniques needed in the analysis of quantal response data.

2. Frequency Distribution of Quantal Response

In quantal assays, occurrence or non-occurrence will depend upon the intensity of the

stimulus. For any one subject, under controlled conditions, there will be a certain level of

intensity below which the response does not occur and above which the response occurs.

Such a value has often been called a threshold or limen, but the term tolerance is now

widely accepted. This tolerance value will vary from one member to another of the

population used, frequently between quite wide limits. When the characteristic response is

quantitative, the stimulus intensity needed to produce a response of any given magnitude

will show similar variation between individuals. In either case, the value for an individual

also is likely to vary from one occasion to another as a result of uncontrolled internal or

external condition.

For quantal response data it is therefore necessary to consider the distribution of

tolereances over the population studied. If the dose, or intensity of the stimulus, is

measured by , the distribution of tolerance may be expressed by

dP = f() d; ...(2.1)

This equation states that a proportion, dp, of the whole population consists of individuals

whose tolerance lie between and +d, where d represents a small interval on the dose

scale, and that dP is the length of this interval multiplied by the appropriate value of the

distribution function f().

If a dose

0

is given to the whole population, all individuals will respond whose tolerances

are less than

0

, and the proportion of these is P, where

Probit Analysis

2

P =

0

0

) (

d f ;

The measure of dose is here assumed to be a quantity that can conceivably range from

zero to +.

Distribution of tolerances, as measured on the natural scale, may be markedly skew, but it

is often possible, by a simple transformation of the scale of measurement, to obtain a

distribution which is approximately normal. The transformation

x = log

10

generally brings normality in the response variable, however, for some fungicides a better

transformation may be

x =

i

, where usually i 1 .

3. Effective Dose

In these assays, the earlier attempts were made to characterize the effectiveness of a

stimulus in relation to a quantal response referred to the minimal effective dose, or, for a

more restricted class of stimuli, the minimal lethal dose, terms, which failed to take

account of the variation in tolerance within a population. The logical weakness of such

concepts is the assumption that there is a dose for any given chemical, which is only just

sufficient to kill all or most of the animals of a given species, and that doses a bit lesser

would not kill any animal of that species. Any worker, however, accustomed to the

estimation of toxicity knows that these assumptions do not represent the truth.

It might be thought that the minimal lethal dose of a poison could instead be defined as the

dose just sufficient to kill a member of the species with the least possible tolerance, and

also a maximal non-lethal dose as the dose, which will just fail to kill the most resistant

member. Undoubtedly some doses are so low that no test subject will succumb to them

and others so high as to prove fatal at all, but considerable difficulties attend

determination of the end-points of these ranges. Even when the tolerance of an individual

can be measured directly, to say, from measurements on a sample of ten or a hundred that

the lowest tolerance found indicated the minimal lethal dose would be unwise; a larger

sample might contain a more extreme member. When only quantal responses for selected

doses can be recorded the difficulty is increased, and the occurrence of exceptional

individuals in the batches at different dose levels may seriously bias the final estimates.

The problem is, in fact, that of determining the dose at which the dose response curve for

the whole population needs the 0% or 100% levels of kill and even a very large

experiment could scarcely estimate these points with any accuracy.

An escape from the dilemma can be made by giving attention to a different and more

satisfactorily defined characteristics, the median lethal dose, or, as a more general term to

include response other then death, the median effective dose. This is the dose that will

produce a response in half the population. The median effective dose is commonly

referred to as the ED 50, the more restricted concept of median lethal dose as the LD 50.

Analogous symbols were used for doses effective for other proportions of the population,

ED 90 being the dose that causes 90% to respond. With a fixed total number of subject

Probit Analysis

3

effective doses in the neighborhood of ED 50 can usually be estimated more precisely

then those for more extreme percentage levels and this is, therefore, particularly favoured

in expressing the effectiveness of the stimulus. The ED 50 alternatively be regarded as the

median of the tolerance distribution that is to say the level of tolerance such that exactly

half the subject lie on either side of it.

For any distribution of tolerance, the ED 50, , satisfies the equation

5 . 0 ) (

0

=

d f .

When a simple normalizing transformation for the doses is available, so that x, the

normalizing measure of dosage, has a normally distributed tolerance, equation (2.1) is

transformable to

dx e dP

x

2

2

) (

2

1

2

1

= , ...(3.1)

where is the center of the distribution and

2

its variance. is the population value of

the mean dosage tolerance, or median effective dosage, and efforts must be directed at

estimating it from the observational data. For the present case the transformation is

logarithmic, so that is the log ED 50; the results obtained are in the main true for any

other transformation, at least as far as they relate to the measure of dosage, x, but

modifications are required in transforming back from x to the scale.

The ED50 alone does not fully describe the effectiveness of the stimulus. Two

insecticides/fungicides may require the same rate of application in order to be lethal to

half of the population, but, if the distribution of tolerances has a lesser 'spread' for one

than for the other, any increase or decrease from this rate will produce a greater change in

mortality for the first than for the second. This spread is measured by the variance,

2

,

the smaller the value of

2

, the greater is the effect on mortality of any change in dose.

Stimuli which produce their effects by similar means, often have approximately equal

variances of their log tolerances for any given population of test subjects, even though

they differ substantially in their median lethal doses. An assessment of the relative

potencies can then be made from median doses alone.

4. Probit Analysis

The ED 50 or LD 50 can easily be calculated using the Probit Analysis. The form of

analysis now used to estimate the parameters and

2

of the distribution of tolerances, is

generally based upon the probit transformation of the experimental results. For doing this,

we conduct an experiment on different doses of an insecticide applied under standardized

conditions to samples of an insect species and record the number of insects killed and the

number of insects exposed. Now the ratio of the number insects died to that of the number

of insects exposed gives the probability of the insects killed at a particular dose. Now this

probability data is subjected to probit transformation that is nothing but the 5 more than

the normal equivalent deviate(this is done to simplify the arithmetical procedure by

avoiding negative values). The probit of the proportion P is defined as the abscissa which

corresponds to a probability P in a normal distribution with mean 5 and variance 1; in

symbols, the probit of P is Y, where

Probit Analysis

4

du e P

Y

u

=

5

2

1

2

2

1

. ...(4.1)

The expected proportion of insects killed by a dosage x

0

is

dx e P

x

x

=

0

2

2

) (

2

1

2

1

. ...(4.2)

Comparison of two formulae for P then shows that the probit of the expected proportion

killed is related to the dosage by the linear equation

Y = 5+ ) (

1

x . ...(4.3)

By means of probit transformation, experimental results may be used to give an estimate

of this equation, and the parameters of the distribution may then be estimated; in

particular, the median effective dosage is estimated as that value of x which gives Y = 5.

Note: The choice of an efficient experimental design is based on the nature of the

variability in the experimental material, environmental conditions and objectives for

conducting a bioassay. The design may be a randomized complete block design, an

incomplete block design, design for factorial experiments etc.

5. Estimation of Parameters

When experimental data on the relationship between dose and mortality have been

obtained, either a graphical or an arithmetical process can be used to estimate the

parameters. Both depend on the probit transformation. The graphical approach is much

more rapid and is sufficiently good for many purposes, but for some, more complex

problems, or when an accurate assessment of the precision of estimates is wanted, the

more detailed arithmetical analysis is necessary.

In order to make either type of estimate, the percentage kill observed for each dose must

first be calculated and converted to probits. The probits are then plotted against x, the

logarithm of the dose and straight line is drawn by eye to fit the points as satisfactorily as

possible. In drawing the line and judging its agreement with the data, only the vertical

deviations of the points must be considered. The line in statistical terminology, the

weighted regression line of the mortality probit on x.

Now LD50 is estimated from the line as m, the dosage at which Y = 5. The slope of the

line, b, which is an estimate of 1/

x. These two parameters are then substituted in equation (4.3) to give the estimated

relationship between dosages and kill. To test whether the line is an adequate

representation of the data, a

2

test may be used. A value of

2

within the limits of

random variation indicates satisfactory agreement theory (the line) and observation (the

data).

Probit Analysis

5

Example 5.1: Table 5.1 contains the data on effect of a series of concentrations of

rotenone when spraying on Macrosiphoniella sanborni, the chrysanthemum aphis, in

batches of about fifty.

Table 5.1: Toxicity of Retenone to Macrosiphoniella sanborni

Concentratio

n (mg./1.)

No. of

insects (n)

No. of

affected

%kill (p) Log

concentration (x)

Empirical

probit

10.2 50 44 88 1.01 6.18

7.7 49 42 86 0.89 6.08

5.1 46 24 52 0.71 5.05

3.8 48 16 33 0.58 4.56

2.6 50 6 12 0.41 3.82

0 49 0 0 - -

Now the probits are plotted against dosage. They almost lie on a straight line. All

predictions can be made from the probit diagram. In the present example a probit value of

5 is given by a dosage of m =0.687; this therefore is the estimate of log LD50, and the

LD50 is estimated as a concentration of 4.86mg/l. Similarly the logLD90 corresponds to a

probit of 6.28 and is therefore and is therefore 1.003; the LD90 is thus estimated as 10.1

mg/l. Again an increase of 0.8 in dosage is associated with an increase of 3.21 in the

probit. Hence the estimated regression coefficient of probit on dosage, or the rate of

increase of probit value per unit increase in x, is

b =1/s = 4.01,

where s (=0.25) is an estimate of

tolerances. The relationship between probit and dosage may be written

Y = 5 + 4.01 (x 0.687), or Y = 2.25 + 4.01x ...(5.1)

Equation (5.1) may be used to calculate expected number of insects killed at each

concentration. By substitution of the values of x used in the experiment, the equation

gives the values of Y. In Table 5.2 expected probits are calculated. Thus a probit of 6.30

corresponds to a percentage of between 90 and 91, or, more accurately, 90+2/6%. If the

expected proportion for any concentration is multiplied by n, the number of insects tested

at that concentration, the result is the expected number of affected insects, or the average

number which would be affected in a batch of size n if equation (5.1) represented the true

relationship between dosage and kill. These numbers, np, may then be compared with the

actual numbers affected, r, in order to judge the adequacy of the equation.

Table 5.2: Comparison of Observed and Expected Mortality.

No. affected Log

concentration

(x)

Y P No. of

insects

(n)

Observed

(r)

Expected

(nP)

Discrepancy

(r-np)

) 1 (

) (

2

P nP

nP r

1.01 6.30 90.3 50 44 45.2 -1.2 0.33

0.89 5.83 79.7 40 42 39.1 2.9 1.06

0.71 5.10 54.0 46 24 24.8 -0.8 0.06

0.58 4.58 33.7 48 16 16.2 -0.2 0.00

0.41 3.90 13.6 50 6 6.8 -0.8 0.11

56 . 1

2

] 3 [

=

Probit Analysis

6

A test of significance of the discrepancies may be obtained by squaring each, dividing the

square by (1-P), and again dividing by the tabulated value nP. The sum of these quantities

is, to a sufficiently close approximation if the line has been well drawn. A

2

; the degree

of freedom are two less than the number of concentrations tested. The value of

2

is 1.56,

which, being less than the table value, is clearly sufficiently small to be attributed to

random fluctuations about the relationship specified in (4.3). Thus the regression line

obtained appears to be a very satisfactory representation of the results of the experiment.

When a parameter such as the median lethal dose has been estimated from experimental

data it is natural to wish to infer within what limits its true value may reasonably be

expected to lie. A statement about the probability, by the use of which probabilities can be

assigned only to statements about the occurrence of observations or of statistics calculated

from the observations. In order to overcome this difficulty, the concept of fiducial

probability has been introduced. These limits can easily be calculated after obtaining the

standard errors of the LD values. In the present example the fiducial limits of LD 50 are m

= 0.687 0.023.

Besides knowing the ED 50 of a particular chemical preparation, the experimenter may be

interested in comparing the relative potencies of the several chemical preparations. One is

required to fit the probit regression lines for each of the chemical preparations separately.

These regression lines are required to be tested for parallelism. If the probit regression

lines are parallel for the different chemical preparations, then the relative potency is

constant at all levels of the response.

Note: Empirical probits can be obtained using Table. This Table is available in Statistical

Tables for Biological, Agricultural and Medical Research by Fisher and Yates (as Table

IX). There are also other methods available to obtain the probit regression line.

(This note is prepared from the book Probit Analysis by D. J. Finney)

EXERCISE

The following data represent the level of concentrations of certain insecticide, number of

insects exposed to the concentrations and number of insects died respectively. Fit a

probit model, obtain the probits for each concentration and plot them. Also obtain LD50

value of the insecticide.

Level of Concentration No. of Insects Tested No. of Insects Died

9.21 376 0

10.21 200 0

10.58 93 0

10.83 120 2

11.08 90 2

11.33 88 5

11.58 105 10

Probit Analysis

7

11.83 111 17

12.08 100 16

12.33 93 29

12.58 100 39

12.83 108 51

13.08 99 47

13.33 106 67

13.58 105 81

13.83 117 88

14.08 98 79

14.33 97 90

14.58 120 113

14.83 102 95

15.08 122 117

15.33 111 107

15.58 94 92

15.83 114 112

17.58 1049 1049

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