LC 50 (Lethal Concentration) SEBUAH PENDEKATAN DALAM UJI TOKSISITAS KUANTITATIF
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Transcript of LC 50 (Lethal Concentration) SEBUAH PENDEKATAN DALAM UJI TOKSISITAS KUANTITATIF
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LC50 (Lethal Concentration) SEBUAH PENDEKATAN DALAM
UJI TOKSISITAS KUANTITATIF
Faculty of Fisheries and Marine SciencesUniversity of Padjadjaran
2009
UJI TOKSISITAS KUANTITATIF (Part 2)
MODULE :
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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?
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Why LC50?
Concentration (or Dose)
Res
pons
e
Low High
0%
100%
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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
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Preparation of Toxicity Test Organisms
Use of test species based on :– Lab hardiness– Common– Known life cycle– Cheap– Short-lived
1
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Use an aquarium for fish or shrimp
Use a vial glass bottle for Dhapnia or Brine Shrimp
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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)
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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
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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
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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
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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
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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)
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Normal distribution of resistance/sensitivityFr
eque
ncy
5% allowable impact
0
10
0
Mean response
Protected
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Preparing Data Tabulation
Format Tabulasi Data Analisis Probit LC50Jenis 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
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Log Probit6.181.009
6.18
1.009
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b = …………………….. Persamaan (2)
a = ……………………. Persamaan (3)
m = ……………………. Persamaan (1)
Determine a,b and m value to determine LC50 value
LC50 = anti Log m = ………………… ppm
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Tolerance
Dose-Response
No.
of I
ndiv
idua
ls
No.
of I
ndiv
idua
ls
Cum
ulat
ive
Perc
ent
Cum
ulat
ive
Perc
ent
Log of Concentration
Log of Concentration
Concentration
Concentration
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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
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2 Metode Matsumura (1985)
Log LC84= 0.93; LC84= 8.51Log LC16 = 0.43; LC16 = 2.69Log LC50 = 0.68; LC50 = 4.79
“ Determination Inverse Prediction”
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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
2S = Log10 (S) =
+
.93 8.51.43 2.69.68 4.79
+8.514.79
4.792.69
2=
1.78 0.250
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3. Determine NN = 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. 2.77Log10(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 = 5.474.79 / 1.143 = 4.19
1.143
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Log scale
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3 Validasi dengan Program Probit
Program Komputasi Perhitungan Nilai Probit :
1. EPA Probit Versi 1.5
2. Micro Probit 3.0
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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 ?
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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)
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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
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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)
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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
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Photo by R. Grippo
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