MODULE :

UJI TOKSISITAS KUANTITATIF (Part 2)

LC50 (Lethal
Concentration)
SEBUAH PENDEKATAN DALAM
UJI TOKSISITAS KUANTITATIF

Faculty of Fisheries and Marine Sciences

University of Padjadjaran
2009

What does LC50 mean?

LC stands for "Lethal Concentration". LC values
usually refer to the concentration of a chemical in
air but in environmental studies it can also mean
the concentration of a chemical in water (Canadian
Centre for Occupational Health and Safety/ CCOHS 2005)

Suatu bahan kimia dinyatakan berkemampuan

toksik akut bila aksi langsungnya mampu
membunuh 50% atau lebih populasi uji dalam
selang waktu yang pendek, misal 24 jam, 48 jam
s/d 14 hari (Meyer et.al. 1982)

Why LC50?

Response

100%

0%
Low

Concentration (or Dose)

High

Preparation for Conducting LC50 Test

1. Organisme Uji
Artemia, Dhapnia : Acute Test
Ikan Mas, Udang : Acute, Sub-Lethal

2. Konsentrasi Uji (I,ii,ii g/mL)

3. Format Tabulasi Data dan Analisis Probit
- Tabel Data Kelompok
- Tabel Data Kelas
- Tabel Rekap Probit

Preparation of Toxicity Test Organisms

Use of test species based on :

Lab hardiness
Common
Known life cycle
Cheap
Short-lived

Use an aquarium for fish or shrimp

Use a vial glass bottle for Dhapnia or Brine Shrimp

Run a dilution series of Toxicant

Test Material (toxicant) conditions :

Pure
Commercial formulation
Mixtures of known concentration
Carriers/solvents
Unknown mixtures (eg. sediment, effluent)

And it must be served in variation of concentration (or Dose)

Preparation of Stock Solution

V1.N1 = V2.N2
x.1000 = 100.100

PA

PS

1000 g/mL

Diambil 10 mL
+ 90 mL medium

Original Concentration

PA

PS

Larutan Stock @ 100 g/mL sebanyak 100 mL

Preparation of Dilution Series

V1.N1 = V2.N2
x.100 = 10.10

100 g/mL
Diambil 1 mL
+ 9 mL medium
Larutan Stock
10 g/mL

Diambil 1 mL
+ 9 mL medium

10 -1

V1.N1 = V2.N2
x.10 = 10.1

1 g/mL
Diambil 1 mL+
9 mL medium

10 -2

0,1 gL
10 -3

V1.N1 = V2.N2
x.1 10. (0,1)

1.
2.
3.
4.

Expose to The Test Medium

Toxicity Test Organisms

10 -1

10 -2

Static Test
Renewable Test
Resirculation Test
Flow-Trough Test

10 -3

Dilution series of Toxicant

Observe Mortality
for Determine LC50

Determine LC50 Using Probit Analysis

Metode Hubert (1979)

Probit = probability unit

% of population responding as a function of
standard deviation units from the mean

Nilai Probit dari persentase mortalitas

merupakan fungsi linear : Y = a + bx

hewan

uji

Nilai LC50 diperoleh dari anti log m

(m merupakan logaritma konsentrasi klorin pada Y = 5/ nilai Probit 50%
hewan uji)
m=
b

5a
. (1)

b = XY 1/ n ( X Y)
X2 1/ n ( X)2 . (2)

a = 1/ n (Y b X) ..(3)

100

Normal distribution of resistance/sensitivity

Frequency

Mean response

Protected

5% allowable
impact

Preparing Data Tabulation

Lihat di Tabel
Probit % Mortalitas

Format Tabulasi Data Analisis Probit LC50

Jenis Hewan Uji
:
Jenis Bahan Toksik
:
d
(Konsentrasi
Uji)

n
(jumlah
hewan uji)

30

ii

30

iii

30

r
(Mortalitas
hewan uji)

Jumlah ( )

p
(%Mortalitas)

X
(Log
Konsentrasi)

Y
(Nilai Probit %
Mortalitas)

XY

XY

Log
1.009

Probit
6.18

6.18

1.009

Determine a,b and m value to determine LC50 value

b = .. Persamaan (2)

a = . Persamaan (3)

m = . Persamaan (1)

LC50 = anti Log m = ppm

No. of Individuals

No. of Individuals

Tolerance

Log of Concentration

Concentration

Cumulative Percent

Cumulative Percent

Dose-Response

Concentration

Log of Concentration

In Probit Analysis we cannot measure normal distribution directly

because effect is cumulative, so graph as cumulative distribution

Cumulative distribution

# Responding

Normal distribution

Dose

Log Dose

Metode Matsumura (1985)

Determination Inverse Prediction

Log LC84= 0.93; LC84= 8.51

Log LC16 = 0.43; LC16 = 2.69
Log LC50 = 0.68; LC50 = 4.79

Calculating 95% Confidence Limits of LC50's

(source: F. Matsumura. 1985. Toxicology of Insecticides, 2nd Ed., Plenum, pp.14-16)
Example Worksheet - Rotenone toxicity to Macrosphoniella sanborni
1. Use inverse prediction from the graph to estimate the Log 10 of the LC84, LC16,
and LC50 then un-log the values and express as mg/L.
Log10 Conc.
Conc. (mg/L)
eg:
LC84 =
8.51
.93
LC16 =
.43
2.69
LC50 =
.68
4.79
2. Calculate S and Log10 (S) (use the 'un-logged' dose/conc. values).
LC84
S = LC50
S=

LC50
LC16
2
Log10 (S)
=
1.78

8.51
4.79

+
2

0.250

4.79
2.69

3. Determine N
N = the total number of individuals tested between the range of dosages that
correspond to the LC16 to the LC84.
N=

49 + 46 + 48 = 143

4. Calculate Log10(f) and f.

2.77
Log10(f) = N x Log10 (S)
Log10(f) =

0.058f =

2.77

x 0.250

143

1.143

5. Calculate Upper and Lower 95% Confidence Limits (multiply or divide 'unlogged'
conc/dose values by f):
Upper Limit = LC50 x f =
Lower Limit = LC50 / f =

4.79 x 1.143 = 5.47

4.79 / 1.143 = 4.19

LC50 = 4.79 mg/L (4.19 5.47; 95% C.L.)

Log scale

Validasi dengan Program Probit

Program Komputasi Perhitungan Nilai Probit :
1. EPA Probit Versi 1.5
2. Micro Probit 3.0

EPA Probit Version 1.5

Case Sample :
Data Mortalitas Larva Udang Windu Setelah Pemaparan 96 jam pada Logam Cu
d (konsentrasi uji, ppm)

n (jumlah hewan uji)

r (mortalitas hewan uji)

0 (Kontrol)

30

0.251

30

0.63

30

1.58

30

13

3.97

30

19

9.96

30

30

Berapa LC50-96 jam ?

EPA Probit Analysis

Report (Out put)

EPA PROBIT ANALYSIS PROGRAM

USED FOR CALCULATING LC/EC VALUES
Version 1.5
LC_Cu_UdangWindu
Proportion
Observed Responding Predicted
Number Number Proportion Adjusted for Proportion
Conc. Exposed Resp. Responding Controls
Responding
0.2510
0.6300
1.5800
3.9700
9.9600

30
30
30
30
30

0
3
13
19
30

0.0000
0.1000
0.4333
0.6333
1.0000

0.0000
0.1000
0.4333
0.6333
1.0000

0.0083
0.0859
0.3674
0.7553
0.9572

Chi - Square for Heterogeneity (calculated) = 4.643

Chi - Square for Heterogeneity
(tabular value at 0.05 level)
= 7.815
Mu
Sigma

=
=

0.330260
0.388480

Parameter
Estimate Std. Err.
95% Confidence Limits
--------------------------------------------------------------------Intercept
4.149867 0.176426 ( 3.804072, 4.495663)
Slope
2.574135 0.344351 ( 1.899208, 3.249063)
Theoretical Spontaneous Response Rate = 0.0000

EPA Probit Analysis

Report (Out put)

LC_Cu_UdangWindu
Estimated LC/EC Values and Confidence Limits
Point

LC50 Value

LC/EC 1.00
LC/EC 5.00
LC/EC 10.00
LC/EC 15.00
LC/EC 50.00
LC/EC 85.00
LC/EC 90.00
LC/EC 95.00
LC/EC 99.00

Exposure
Conc.
0.267
0.491
0.680
0.847
2.139
5.406
6.732
9.317
17.139

95% Confidence Limits

Lower
Upper
0.122
0.274
0.419
0.555
1.665
4.007
4.837
6.355
10.490

0.431
0.712
0.936
1.132
2.762
8.407
11.154
17.060
38.270

EPA Probit Analysis

Report (Out put)

LC_Cu_UdangWindu
PLOT OF ADJUSTED PROBITS AND PREDICTED REGRESSION LINE
Probit
7+
.
..
..
...
....
6+
....
....
...
.... o
....
5+
...
o....
....
...
....
4+
....
o....
...
..
..
3+ .
-.
2+
1+
-+--------------+--------+---------+---------+--------+--------------+EC01
EC10 EC25
EC50
EC75 EC90

EC99

Take Home Task!

Determine LC50-96hr Value from the Case

Sample using Manual Probit Analysis Hubert
(1979) Metodh!
And please attach also EPA Probit V 1.5 Report
(Out put sheet) as validation

Deadline
: Nov 5,
2009

Thank you

If there is magic on earth, it is in water

Photo by R. Grippo