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

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)

Why LC50? Concentration (or Dose) Response 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 1 Preparation of Toxicity Test Organisms Use of test species based on : Lab hardiness Common Known life cycle Cheap Short-lived

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

Run a dilution series of Toxicant 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)

Preparation of Stock Solution PS H Original Concentration V1.N1 = V2.N2 x.1000 = 100.100 1000 µg/mL Diambil 10 mL + 90 mL medium Larutan Stock @ 100 µg/mL sebanyak 100 mL PA PS H

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.1 10. (0,1) Diambil 1 mL+ 9 mL medium 10 -2 10 -3

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

Determine LC50 Using Probit Analysis 3 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

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)

Normal distribution of resistance/sensitivity Frequency 5% allowable impact 0 100 Mean response Protected Resistance (log [X]

(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

Log Probit 6.18 1.009 6.18 1.009

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

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

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

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

+ + 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 2.69 2 = 1.78 0.250

= 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 = = 49 + 46 + 48 = 143 2.77 x 0.250  143 0.058 1.143 4.79 x 1.143 = 5.47 4.79 / 1.143 = 4.19 LC50 = 4.79 mg/L (4.19 – 5.47; 95% C.L.)

Log scale

Validasi dengan Program Probit 3 Validasi dengan Program Probit Program Komputasi Perhitungan Nilai Probit : EPA Probit Versi 1.5 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 0.251 0.63 3 1.58 13 3.97 19 9.96 Berapa LC50-96 jam ?

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 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 = 0.330260 Sigma = 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 Limits Point Conc. Lower Upper LC/EC 1.00 0.267 0.122 0.431 LC/EC 5.00 0.491 0.274 0.712 LC/EC 10.00 0.680 0.419 0.936 LC/EC 15.00 0.847 0.555 1.132 LC/EC 50.00 2.139 1.665 2.762 LC/EC 85.00 5.406 4.007 8.407 LC/EC 90.00 6.732 4.837 11.154 LC/EC 95.00 9.317 6.355 17.060 LC/EC 99.00 17.139 10.490 38.270 LC50 Value

EPA Probit Analysis Report (Out put) 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 : Nov 5, 2009

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