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 environ ID: 729334
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Slide1
LC
50 (Lethal Concentration) SEBUAH PENDEKATAN DALAM UJI TOKSISITAS KUANTITATIF
Faculty of Fisheries and Marine SciencesUniversity of Padjadjaran2009
UJI TOKSISITAS KUANTITATIF (Part 2)
MODULE :Slide2
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?Slide3
Why LC50?
Concentration (or Dose)
Response
Low
High
0%
100%Slide4
Preparation for Conducting LC50 Test
1. Organisme Uji Artemia, Dhapnia : Acute Test Ikan Mas, Udang : Acute, Sub-Lethal2. Konsentrasi Uji (I,ii,ii µg/mL
) 3. Format Tabulasi Data dan Analisis Probit - Tabel Data Kelompok
- Tabel Data Kelas - Tabel Rekap ProbitSlide5
Preparation of Toxicity Test Organisms
Use of test species based on :Lab hardinessCommonKnown life cycleCheapShort-lived
1Slide6
Use an aquarium for fish or shrimp
Use a vial glass bottle for Dhapnia or Brine ShrimpSlide7
2
Run a dilution series of Toxicant
Test Material (toxicant) conditions : PureCommercial formulationMixtures of known concentration
Carriers/solventsUnknown mixtures (eg. sediment, effluent)
And it must be served in variation of concentration (or Dose)Slide8
PA
PS
H
Original Concentration
1000 µg/mL
V1.N1 = V2.N2
x.1000 = 100.100
Diambil 10 mL
+ 90 mL medium
Larutan Stock @ 100 µg/mL sebanyak 100 mL
PA
PS
H
Preparation of Stock SolutionSlide9
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.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 SeriesSlide10
Dilution series of Toxicant
10 -
2
10 -
1
10 -
3
Toxicity Test Organisms
Static Test
Renewable Test
Resirculation Test
Flow-Trough Test
Observe Mortality for Determine LC
50
Expose to The Test MediumSlide11
Metode Hubert (1979)
Probit = probability unit% of population responding as a function of standard deviation units from the meanNilai Probit dari persentase mortalitas hewan uji merupakan fungsi linear : Y = a + bx
3
Determine LC50 Using Probit Analysis1Slide12
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)Slide13
Normal distribution of resistance/sensitivity
Resistance (log [X]
Frequency
5% allowable impact
0 100
Mean response
ProtectedSlide14
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 % MortalitasSlide15
Log
Probit
6.18
1.009
6.18
1.009Slide16
b = …………………….. Persamaan (2)a = ……………………. Persamaan (3)
m = ……………………. Persamaan (1)Determine a,b and m value to determine LC50 value
LC50 = anti Log m = ………………… ppmSlide17
Tolerance
Dose-Response
No. of Individuals
No. of Individuals
Cumulative Percent
Cumulative Percent
Log of Concentration
Log of Concentration
Concentration
ConcentrationSlide18
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 distributionSlide19
2
Metode Matsumura (1985)
Log LC84
= 0.93; LC84= 8.51
Log LC16 = 0.43; LC
16 = 2.69
Log LC50
= 0.68; LC50
= 4.79
“ Determination Inverse Prediction”Slide20
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
0.250Slide21
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
1.143Slide22Slide23
Log scaleSlide24
3
Validasi dengan Program ProbitProgram Komputasi Perhitungan Nilai Probit :EPA Probit Versi 1.5Micro Probit 3.0Slide25
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 ?Slide26
EPA PROBIT ANALYSIS PROGRAMUSED FOR CALCULATING LC/EC VALUES Version 1.5LC_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 = 0.330260Sigma = 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) Slide27
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 0.431LC/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 ValueSlide28
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) Slide29
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, 2009Slide30
Photo by R. Grippo
If there is magic on earth, it is in waterThank you