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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
2012

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?

Response

100%

0%

Low

Concentration (or Dose)

High

Preparation for Conducting LC50 Test


1. Organisme Uji
Artemia, Dhapnia : Acute Test
Ikan Mas, Udang : Acute, Sub-Lethal

2. Konsentrasi Uji (I,ii,ii g/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

Use an aquarium for fish or shrimp

Use a vial glass bottle for Dhapnia or Brine Shrimp

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

1000 g/mL

Diambil 10 mL
+ 90 mL medium

Original Concentration

PA

PS

Larutan Stock @ 100 g/mL sebanyak 100 mL

Preparation of Dilution Series

V1.N1 = V2.N2
x.100 = 10.10

100 g/mL
Diambil 1 mL
+ 9 mL medium
Larutan Stock

10 g/mL
Diambil 1 mL
+ 9 mL medium

10 -1

V1.N1 = V2.N2
x.10 = 10.1

1 g/mL

Diambil 1 mL+
9 mL medium

10 -2

0,1 gL
10 -3

V1.N1 = V2.N2
x.1 10. (0,1)

1.
2.
3.
4.

Expose to The Test Medium

Toxicity Test Organisms

10 -1

10 -2

Static Test
Renewable Test
Resirculation Test
Flow-Trough Test

10 -3

Dilution series of Toxicant

Observe Mortality
for Determine LC50

Determine LC50 Using Probit Analysis

Metode Hubert (1979)

Probit = probability unit


% of population responding as a function of
standard deviation units from the mean

Nilai Probit dari persentase mortalitas


merupakan fungsi linear : Y = a + bx

hewan

uji

Nilai LC50 diperoleh dari anti log m


(m merupakan logaritma konsentrasi klorin pada Y = 5/ nilai Probit 50%
hewan uji)
m=

5a
b

. (1)

b = XY 1/ n ( X Y)
X2 1/ n ( X)2

. (2)

a = 1/ n (Y b X)

..(3)

100

Normal distribution of resistance/sensitivity

Frequency

Mean response

Protected

5% allowable
impact

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)

30

ii

30

iii

30

r
(Mortalitas
hewan uji)

Jumlah ( )

p
(%Mortalitas)

X
(Log
Konsentrasi)

Y
(Nilai Probit %
Mortalitas)

XY

XY

Log
1.009

Probit
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

Tolerance

Log of Concentration

Concentration

Dose-Response

Concentration

Log of Concentration

In Probit Analysis we cannot measure normal distribution directly


because effect is cumulative, so graph as cumulative distribution

Cumulative distribution

# Responding

Normal distribution

Dose

Log Dose

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

0.251

30

0.63

30

1.58

30

13

3.97

30

19

9.96

30

30

Berapa LC50-96 jam ?

EPA PROBIT ANALYSIS PROGRAM


USED FOR CALCULATING LC/EC VALUES
Version 1.5

EPA Probit Analysis


Report (Out put)

LC_Cu_UdangWindu
Proportion
Observed Responding Predicted
Number Number Proportion Adjusted for Proportion
Conc. Exposed Resp. Responding Controls
Responding
0.2510
0.6300
1.5800
3.9700
9.9600

30
30
30
30
30

0
3
13
19
30

0.0000
0.1000
0.4333
0.6333
1.0000

0.0000
0.1000
0.4333
0.6333
1.0000

0.0083
0.0859
0.3674
0.7553
0.9572

Chi - Square for Heterogeneity (calculated) = 4.643


Chi - Square for Heterogeneity
(tabular value at 0.05 level)
= 7.815
Mu
Sigma

=
=

0.330260
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

Point

LC50 Value

LC/EC 1.00
LC/EC 5.00
LC/EC 10.00
LC/EC 15.00
LC/EC 50.00
LC/EC 85.00
LC/EC 90.00
LC/EC 95.00
LC/EC 99.00

Exposure
Conc.

0.267
0.491
0.680
0.847
2.139
5.406
6.732
9.317
17.139

95% Confidence Limits


Lower
Upper

0.122
0.274
0.419
0.555
1.665
4.007
4.837
6.355
10.490

0.431
0.712
0.936
1.132
2.762
8.407
11.154
17.060
38.270

LC_Cu_UdangWindu

EPA Probit Analysis


Report (Out put)

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

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

Thank you

If there is magic on earth, it is in water

Photo by R. Grippo