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LC 50 (Lethal Concentration) SEBUAH PENDEKATAN DALAM UJI TOKSISITAS KUANTITATIF Faculty of Fisheries and Marine Sciences University of Padjadjaran 2009.

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Presentation on theme: "LC 50 (Lethal Concentration) SEBUAH PENDEKATAN DALAM UJI TOKSISITAS KUANTITATIF Faculty of Fisheries and Marine Sciences University of Padjadjaran 2009."— Presentation transcript:

1 LC 50 (Lethal Concentration) SEBUAH PENDEKATAN DALAM UJI TOKSISITAS KUANTITATIF Faculty of Fisheries and Marine Sciences University of Padjadjaran 2009 UJI TOKSISITAS KUANTITATIF (Part 2) MODULE :

2 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?

3 Why LC 50 ? Concentration (or Dose) Response LowHigh 0% 100%

4 Preparation for Conducting LC 50 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 Organisms Use of test species based on : –Lab hardiness –Common –Known life cycle –Cheap –Short-lived 1

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

7 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)

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

9 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 = V1.N1 = V2.N2 x.10 = 10.1 V1.N1 = V2.N2 x (0,1) Preparation of Dilution Series

10 Dilution series of Toxicant Toxicity Test Organisms 1.Static Test 2.Renewable Test 3.Resirculation Test 4.Flow-Trough Test Observe Mortality for Determine LC 50 Expose to The Test Medium

11 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

12 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)

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

14 Preparing Data Tabulation Format Tabulasi Data Analisis Probit LC 50 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 i30 ii30 iii30 Jumlah ( ∑ )∑ X∑Y∑XY Lihat di Tabel Probit % Mortalitas

15 Log Probit

16 b = …………………….. Persamaan (2) a = ……………………. Persamaan (3) m = ……………………. Persamaan (1) Determine a,b and m value to determine LC 50 value LC 50 = anti Log m = ………………… ppm

17 Tolerance Dose-Response No. of Individuals Cumulative Percent Log of Concentration 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 2 Metode Matsumura (1985) Log LC 84 = 0.93; LC 84 = 8.51 Log LC 16 = 0.43; LC 16 = 2.69 Log LC 50 = 0.68; LC 50 = 4.79 “ Determination Inverse Prediction”

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). LC84 LC50 S =LC50 LC16 2 S =Log10 (S) = =

21 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 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.) = x  x = / =

22

23 Log scale

24 3 Validasi dengan Program Probit Program Komputasi Perhitungan Nilai Probit : 1.EPA Probit Versi 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) Berapa LC jam ?

26 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. 95% Confidence Limits Intercept ( , ) Slope ( , ) Theoretical Spontaneous Response Rate = EPA Probit Analysis Report (Out put)

27 LC_Cu_UdangWindu Estimated LC/EC Values and Confidence Limits Exposure 95% Confidence Limits Point Conc. Lower Upper LC/EC LC/EC LC/EC LC/EC LC/EC LC/EC LC/EC LC/EC LC/EC EPA Probit Analysis Report (Out put) LC 50 Value

28 LC_Cu_UdangWindu PLOT OF ADJUSTED PROBITS AND PREDICTED REGRESSION LINE Probit o o o EC01 EC10 EC25 EC50 EC75 EC90 EC99 EPA Probit Analysis Report (Out put)

29 Take Home Task! Determine LC hr 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 Photo by R. Grippo If there is magic on earth, it is in water Thank you


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