1. 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

2. 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)

3. Why LC50?
Concentration (or Dose)
Response
Low
High 0%
100%

4. 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

5. Preparation of Toxicity Test Organisms1
Preparation of Toxicity Test Organisms
Use of test species based on :
Lab hardiness
Common
Known life cycle
Cheap
Short-lived

6. Use an aquarium for fish or shrimp
Use a vial glass bottle for Dhapnia or Brine Shrimp

7. Run a dilution series of Toxicant2
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)

8. Preparation of Stock SolutionPS H
Original Concentration
V1.N1 = V2.N2
x.1000 =
1000 µg/mL
Diambil 10 mL
+ 90 mL medium
Larutan 100 µg/mL sebanyak 100 mL PA PS H

9. Preparation of Dilution Series
Larutan Stock
100 µg/mL
10 µg/mL
1 µg/mL
0,1 µgL
V1.N1 = V2.N2
x.100 = 10.10
Diambil 1 mL
+ 9 mL medium
V1.N1 = V2.N2
x.10 = 10.1
Diambil 1 mL
+ 9 mL medium
10 -1
V1.N1 = V2.N2
x (0,1)
Diambil 1 mL+ 9 mL medium
10 -2
10 -3

10. Expose to The Test Medium
Static Test
Renewable Test
Resirculation Test
Flow-Trough Test
Expose to The Test Medium
Dilution series of Toxicant
10 -2
10 -1
10 -3
Toxicity Test Organisms
Observe Mortality for Determine LC50

11. Determine LC50 Using Probit Analysis3
Determine LC50 Using Probit Analysis 1
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

12. Nilai LC50 diperoleh dari anti log m
(m merupakan logaritma konsentrasi klorin pada Y = 5/ nilai Probit 50% hewan uji)
m = – a
b ……………………. (1)
b = ∑ XY – 1/ n (∑ X ∑Y)
∑ X2 – 1/ n (∑ X)2 ……………………. (2)
a = 1/ n (∑Y – b ∑ X) ……………………..(3)

13. Normal distribution of resistance/sensitivity
Frequency
5% allowable impact
Mean response
Protected
Resistance (log [X]

14. (Mortalitas hewan uji) (Nilai Probit % Mortalitas)
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) r
(Mortalitas hewan uji) p
(%Mortalitas) X
(Log Konsentrasi) Y
(Nilai Probit % Mortalitas) XY i 30 ii
iii
Jumlah ( ∑ )
∑ X ∑Y
∑XY

15. Log
Probit
6.18
1.009
6.18
1.009

16. Determine a,b and m value to determine LC50 value
b = …………………….. Persamaan (2)
a = ……………………. Persamaan (3)
m = ……………………. Persamaan (1)
LC50 = anti Log m = ………………… ppm

17. Tolerance Dose-Response No. of Individuals No. of Individuals
Log of Concentration
Concentration
Dose-Response
Cumulative Percent
Cumulative Percent
Concentration
Log of Concentration

18. In Probit Analysis we cannot measure normal distribution directly because effect is cumulative, so graph as cumulative distribution
Log Dose
Cumulative distribution
Dose
# Responding
Normal distribution

19. Metode Matsumura (1985) “ Determination Inverse Prediction” 2
Log LC84= 0.93; LC84= 8.51
Log LC16 = 0.43; LC16 = 2.69
Log LC50 = 0.68; LC50 = 4.79

20. + + Calculating 95% Confidence Limits of LC50's
(source: F. Matsumura 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).
LC LC50
S = LC LC16 2
S = Log10 (S) = +
.93
8.51
.43
2.69
.68
4.79 +
8.51
4.79
2.69 2 =
1.78
0.250

21. = LC50 = 4.79 mg/L (4.19 – 5.47; 95% C.L.) 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 = =
= 143 x
 143
0.058
1.143
4.79 x = 5.47
4.79 / = 4.19
LC50 = 4.79 mg/L (4.19 – 5.47; 95% C.L.)

23. Log scale

24. Validasi dengan Program Probit3
Validasi dengan Program Probit
Program Komputasi Perhitungan Nilai Probit :
EPA Probit Versi 1.5
Micro Probit 3.0

25. 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
0.63 3
1.58 13
3.97 19
9.96
Berapa LC50-96 jam ?

26. 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
Chi - Square for Heterogeneity (calculated) =
Chi - Square for Heterogeneity
(tabular value at 0.05 level) =
Mu =
Sigma =
Parameter Estimate Std. Err % Confidence Limits
Intercept ( , )
Slope ( , )
Theoretical Spontaneous Response Rate =

27. EPA Probit Analysis Report (Out put)
LC_Cu_UdangWindu
Estimated LC/EC Values and Confidence Limits
Exposure % Confidence Limits
Point Conc Lower Upper
LC/EC
LC/EC
LC/EC
LC/EC
LC/EC
LC/EC
LC/EC
LC/EC
LC/EC
LC50 Value

28. EPA Probit Analysis Report (Out put)
LC_Cu_UdangWindu
PLOT OF ADJUSTED PROBITS AND PREDICTED REGRESSION LINE
Probit - o
o....
o.... -. 2+ 1+
EC EC EC EC EC EC EC99
EPA Probit Analysis Report (Out put)

29. 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

30. Thank you If there is magic on earth, it is in water
Photo by R. Grippo