LC50 (Lethal Concentration) SEBUAH PENDEKATAN DALAM
UJI TOKSISITAS KUANTITATIF
Faculty of Fisheries and Marine SciencesUniversity of Padjadjaran
2009
UJI TOKSISITAS KUANTITATIF (Part 2)
MODULE :
Checked
Substance Quantities (Amount)
Substance Properties
Substance Characteristics
Organisms Tolerance (Bio assay)
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)
Res
pon
se
Low High
0%
100%
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
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 µg/mL
V1.N1 = V2.N2x.1000 = 100.100
Diambil 10 mL+ 90 mL medium
Larutan Stock @ 100 µg/mL sebanyak 100 mL
PA PS H
Preparation of Stock Solution
10 -1
10 -2
10 -3
Larutan Stock
100 µg/mL
10 µg/mL
1 µg/mL
0,1 µgL
Diambil 1 mL+ 9 mL medium
Diambil 1 mL+ 9 mL medium
Diambil 1 mL+ 9 mL medium
V1.N1 = V2.N2x.100 = 10.10
V1.N1 = V2.N2x.10 = 10.1
V1.N1 = V2.N2x.1 10. (0,1)
Preparation of Dilution Series
Dilution series of Toxicant
10 -210 -1 10 -3Toxicity Test Organisms
1. Static Test2. Renewable Test3. Resirculation Test4. Flow-Trough Test
Observe Mortality for Determine LC50
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 – ab ……………………. (1)
b = ∑ XY – 1/ n (∑ X ∑Y) ∑ X2 – 1/ n (∑ X)2 ……………………. (2)
a = 1/ n (∑Y – b ∑ X) ……………………..(3)
Normal distribution of resistance/sensitivityF
requ
ency
5% allowable impact
0
100
Mean response
Protected
Preparing Data Tabulation
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 30
iii 30
Jumlah ( ∑ ) ∑ X ∑Y ∑XY
Lihat di Tabel Probit % Mortalitas
Log Probit
6.181.009
6.18
1.009
b = …………………….. Persamaan (2)
a = ……………………. Persamaan (3)
m = ……………………. Persamaan (1)
Determine a,b and m value to determine LC50 value
LC50 = anti Log m = ………………… ppm
Tolerance
Dose-Response
No.
of
Ind
ivid
ual
s
No.
of
Ind
ivid
ual
s
Cu
mul
ativ
e P
erce
nt
Cu
mul
ativ
e P
erce
nt
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
espo
ndin
g
Normal distribution
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 PROGRAMUSED 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) = 4.643Chi - Square for Heterogeneity (tabular value at 0.05 level) = 7.815 Mu = 0.330260Sigma = 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 LimitsPoint Conc. Lower Upper LC/EC 1.00 0.267 0.122 0.431LC/EC 5.00 0.491 0.274 0.712LC/EC 10.00 0.680 0.419 0.936LC/EC 15.00 0.847 0.555 1.132LC/EC 50.00 2.139 1.665 2.762LC/EC 85.00 5.406 4.007 8.407LC/EC 90.00 6.732 4.837 11.154LC/EC 95.00 9.317 6.355 17.060LC/EC 99.00 17.139 10.490 38.270
EPA Probit Analysis Report (Out put)
LC50 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 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 : Oct 27, 2010
Photo by R. Grippo
If there is magic on earth, it is in water
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