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 µg/mL)
3. Format Tabulasi Data dan Analisis Probit
- Tabel Data Kelompok
- Tabel Data Kelas
- Tabel Rekap Probit
1
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
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)
Preparation of Stock Solution
V1.N1 = V2.N2
x.1000 = 100.100
PA
PS
H
1000 µg/mL
Diambil 10 mL
+ 90 mL medium
Original Concentration
PA
PS
H
Larutan Stock @ 100 µg/mL sebanyak 100 mL
Preparation of Dilution Series
V1.N1 = V2.N2
x.100 = 10.10
100 µg/mL
Diambil 1 mL
+ 9 mL medium
Larutan Stock
10 µg/mL
Diambil 1 mL
+ 9 mL medium
10 -1
V1.N1 = V2.N2
x.10 = 10.1
1 µg/mL
Diambil 1 mL+
9 mL medium
10 -2
0,1 µgL
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
3
1
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=
5–a
b
……………………. (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
0
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)
i
30
ii
30
iii
30
r
(Mortalitas
hewan uji)
Jumlah ( ∑ )
p
(%Mortalitas)
X
(Log
Konsentrasi)
Y
(Nilai Probit %
Mortalitas)
XY
∑X
∑Y
∑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
Tolerance
Log of Concentration
Concentration
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
2
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).
S =
S=
LC84
LC50
8.51
4.79
+
+
LC50
LC16
4.79
2.69
=
2
2
Log10 (S) = 0.250
1.78
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) = 0.058
f=
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 = 4.79 x 1.143 = 5.47
Lower Limit = LC50 / f = 4.79 / 1.143 = 4.19
LC50 = 4.79 mg/L (4.19 – 5.47; 95% C.L.)
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 LC50-96 jam ?
EPA PROBIT ANALYSIS PROGRAM
USED FOR CALCULATING LC/EC VALUES
Version 1.5
EPA Probit Analysis
Report (Out put)
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
LC_Cu_UdangWindu
EPA Probit Analysis
Report (Out put)
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
Thank you
If there is magic on earth, it is in water
Photo by R. Grippo