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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
Substance Quantities (Amount)

Substance Properties

Checked
Substance Characteristics

Organisms Tolerance (Bio assay)


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?

100%
Response

0%

Low High
Concentration (or Dose)
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


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


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

V1.N1 = V2.N2
x.1000 = 100.100

PA PS H

1000 µg/mL Diambil 10 mL


+ 90 mL medium

Original Concentration

PA PS H

Larutan Stock @ 100 µg/mL sebanyak 100 mL


Preparation of Dilution Series

V1.N1 = V2.N2
100 µg/mL
x.100 = 10.10
Diambil 1 mL
+ 9 mL medium
Larutan Stock

10 µg/mL V1.N1 = V2.N2


x.10 = 10.1
Diambil 1 mL
10 -1 + 9 mL medium

1 µg/mL V1.N1 = V2.N2


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

0,1 µgL

10 -3
1. Static Test
Expose to The Test Medium 2. Renewable Test
3. Resirculation Test
4. Flow-Trough Test

Toxicity Test Organisms 10 -1 10 -2 10 -3

Dilution series of Toxicant

Observe Mortality
for Determine LC50
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
100

Mean response
Frequency

Protected
5% allowable
impact
0
Preparing Data Tabulation Lihat di Tabel
Probit % Mortalitas
Format Tabulasi Data Analisis Probit LC50
Jenis Hewan Uji :………………………………………
Jenis Bahan Toksik :………………………………………

d n r p X Y XY
(Konsentrasi (jumlah (Mortalitas hewan (%Mortalitas) (Log Konsentrasi) (Nilai Probit %
Uji) hewan uji) uji) Mortalitas)

i 30

ii 30

iii 30

Jumlah ( ∑ ) ∑X ∑Y ∑XY
Log Probit
1.009 6.18

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


No. of Individuals Tolerance

No. of Individuals
Concentration Log of 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

Normal distribution Cumulative distribution


# Responding

Dose Log Dose


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 EPA PROBIT ANALYSIS PROGRAM
USED FOR CALCULATING LC/EC VALUES
Report (Out put) 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
LC_Cu_UdangWindu
Report (Out put)
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
LC50 Value 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
LC_Cu_UdangWindu
EPA Probit Analysis PLOT OF ADJUSTED PROBITS AND PREDICTED REGRESSION LINE
Report (Out put) Probit
-
7+ .
- ..
- ..
- ...
- ....
6+ ....
- ....
- ...
- .... o
- ....
5+ ...
- o....
- ....
- ...
- ....
4+ ....
- o....
- ...
- ..
- ..
3+ .
-
-.
-
-
2+
-
-
-
-
1+
-+--------------+--------+---------+---------+--------+--------------+-
EC01 EC10 EC25 EC50 EC75 EC90 EC99
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
: Oct 27
, 2010
Thank you

If there is magic on earth, it is in water

Photo by R. Grippo