LC50 (Lethal Concentration) SEBUAH PENDEKATAN DALAM

UJI TOKSISITAS KUANTITATIF

Faculty of Fisheries and Marine SciencesUniversity 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 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

pons

e

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/sensitivityFr

eque

ncy

5% allowable impact

0

10

0

Mean response

Protected

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

Log Probit6.181.009

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 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

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

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”

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

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

Log scale

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 : Nov 5, 2009

Photo by R. Grippo

If there is magic on earth, it is in water

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