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LC

50
(Lethal Concentration)
SEBUAH PENDEKATAN DALAM
UJI TOKSISITAS KUANTITATIF
Faculty of Fisheries and Marine Sciences
University of Padjadjaran
2009
UJI TOKSISITAS KUANTITATIF (Part 2)
MODULE :
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)

What does LC50 mean?
Why LC50?
Concentration (or Dose)
R
e
s
p
o
n
s
e

Low High
0%
100%
Preparation for Conducting LC
50
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
1
Use an aquarium for fish or shrimp
Use a vial glass bottle for Dhapnia or Brine Shrimp
2
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)
PA PS H
Original Concentration
1000 g/mL
V1.N1 = V2.N2
x.1000 = 100.100
Diambil 10 mL
+ 90 mL medium
Larutan Stock @ 100 g/mL sebanyak 100 mL
PA
PS
H
Preparation of Stock Solution
10 -1
10 -2
10 -3
Larutan Stock
100 g/mL
10 g/mL
1 g/mL
0,1 gL
Diambil 1 mL
+ 9 mL medium
Diambil 1 mL
+ 9 mL medium
Diambil 1 mL+
9 mL medium
V1.N1 = V2.N2
x.100 = 10.10
V1.N1 = V2.N2
x.10 = 10.1
V1.N1 = V2.N2
x.1 10. (0,1)
Preparation of Dilution Series
Dilution series of Toxicant
10 -
2
10 -
1
10 -
3
Toxicity Test Organisms
1. Static Test
2. Renewable Test
3. Resirculation Test
4. Flow-Trough Test
Observe Mortality
for Determine LC
50

Expose to The Test Medium
Metode Hubert (1979)


Probit = probability unit
% of population responding as a function of
standard deviation units from the mean


Nilai Probit dari persentase mortalitas hewan uji
merupakan fungsi linear : Y = a + bx

3
Determine LC50 Using Probit Analysis
1
Nilai LC50 diperoleh dari anti log m
(m merupakan logaritma konsentrasi klorin pada Y = 5/ nilai Probit 50%
hewan uji)

m = 5 a
b . (1)


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



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


Normal distribution of resistance/sensitivity
Resistance (log [X]
F
r
e
q
u
e
n
c
y

5% allowable
impact
0























































1
0
0


Mean response
Protected
Preparing Data Tabulation
Format Tabulasi Data Analisis Probit LC
50

Jenis Hewan Uji :
Jenis Bahan Toksik :

d
(Konsentrasi
Uji)
n
(jumlah
hewan uji)
r
(Mortalitas
hewan uji)
p
(%Mortalitas)
X
(Log
Konsentrasi)
Y
(Nilai Probit %
Mortalitas)
XY
i 30
ii 30
iii 30
Jumlah ( ) X Y XY
Lihat di Tabel
Probit % Mortalitas
Log
Probit
6.18
1.009
6.18
1.009
b = .. Persamaan (2)
a = . Persamaan (3)
m = . Persamaan (1)
Determine a,b and m value to determine LC
50
value
LC
50
= anti Log m = ppm
Tolerance
Dose-Response
Log of Concentration
Log of Concentration
Concentration
Concentration
In Probit Analysis we cannot measure normal distribution directly
because effect is cumulative, so graph as cumulative distribution
Log Dose
Cumulative distribution
Dose
#


R
e
s
p
o
n
d
i
n
g

Normal distribution
2 Metode Matsumura (1985)
Log LC84= 0.93; LC84= 8.51
Log LC16 = 0.43; LC16 = 2.69
Log LC50 = 0.68; LC50 = 4.79
Determination Inverse Prediction
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 =
LC16 =
LC50 =

2. Calculate S and Log10 (S) (use the 'un-logged' dose/conc. values).

LC84 LC50
S = LC50 LC16
2
S = Log10 (S) =
+
.93 8.51
.43 2.69
.68
4.79
+
8.51
4.79
4.79
2.69
2
=
1.78 0.250
3. Determine N
N = the total number of individuals tested between the range of dosages that
correspond to the LC16 to the LC84.

N =

4. Calculate Log10(f) and f.
2.77
Log10(f) = N x Log10 (S)

Log10(f) = f =

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 =

=
LC50 = 4.79 mg/L (4.19 5.47; 95% C.L.)
49 + 46 + 48 = 143
2.77 x 0.250
143
0.058
4.79 x 1.143 = 5.47
4.79 / 1.143 = 4.19
1.143
Log scale
3 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
0.251 30 0
0.63 30 3
1.58 30 13
3.97 30 19
9.96 30 30
Berapa LC
50
-96 jam ?
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 30 0 0.0000 0.0000 0.0083
0.6300 30 3 0.1000 0.1000 0.0859
1.5800 30 13 0.4333 0.4333 0.3674
3.9700 30 19 0.6333 0.6333 0.7553
9.9600 30 30 1.0000 1.0000 0.9572


Chi - Square for Heterogeneity (calculated) = 4.643
Chi - Square for Heterogeneity
(tabular value at 0.05 level) = 7.815

Mu = 0.330260
Sigma = 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

Exposure 95% Confidence Limits
Point Conc. Lower Upper

LC/EC 1.00 0.267 0.122 0.431
LC/EC 5.00 0.491 0.274 0.712
LC/EC 10.00 0.680 0.419 0.936
LC/EC 15.00 0.847 0.555 1.132
LC/EC 50.00 2.139 1.665 2.762
LC/EC 85.00 5.406 4.007 8.407
LC/EC 90.00 6.732 4.837 11.154
LC/EC 95.00 9.317 6.355 17.060
LC/EC 99.00 17.139 10.490 38.270


EPA Probit Analysis
Report (Out put)
LC
50
Value
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
EPA Probit Analysis
Report (Out put)
Take Home Task!
Determine LC
50
-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
Photo by R. Grippo
If there is magic on earth, it is in water
Thank you