Document G5g4z8Ggr2jqjzkeOeKDwQ1Bm
AR226-2967
Study Sponsor: Issue Date: Document Reference No.:
ATOCHEM Unavailable (Data Only)
Bacterial Growth Inhibition Author(s): J.C. Boutonnet A.M. Petit
English Translation of Report Summary for DuPont Chemical Solutions Enterprise 27-Jan-OS
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TRANSLATION
ATOCHEM Levallois Applications Center Analysis - Environmental Services Eco-toxicology Laboratory
TEST REPORT
DETERMINATION OF AN INHIBITING EFFECT ON BACTERIAL PSEUDOMONAS PUTIDA CELL GROWTH
according to proposed standard ISO TC147/SC5/WG1/N111
Substance studied:
Name: Customer reference :
Internal sample reference :
Person responsible for test: Person carrying out test:
Date test started: Date report written:
J. C. BOUTONNET A. M. PETIT
22.10.91
Sample preparation
Mother solution at 20 g/l in ultra-pure water
Organism tested Pseudomonas putida MIGULA, STRAIN Berlin 33/2 (DSM 50025)
Test conditions
Equipment:
Sterilized glassware Perkin-Elmer spectrophotometer Lamda 5
Thermostatically controlled agitator at 21 1 C
Concentration range (each concentration is tested in triplicate):
Vol. Substance solution (ml)
15
10
5
2.5
Vol. Water (ml)
65
70
75
77.5
Vol. Medium (ml)
10
10
10
10
10
10
Vol. Inoculum (ml)
10
10
10
10
10
10
Substance concentration (g/l) 3
2
1
0.5
The flasks 'are incubated in a thermostatically controlled agitator
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The culture is started on 22/10/91 at 16 h and stopped on 23/10/91 at 8 h 30 Incubation duration : 16 h 30
The turbidity is measured using a spectrophotometer by determination of the optical density at a wavelength of 436 nm (ISO standard 7027 -1984) after dilution : 10 before incubation
Results
Dilution at 1/10 to effect the spectrophotometer reading
Substance concentration (mg/l) (Optical density of control = 0.0037)
3 2
1
0.5
OD in flask
1
2
0.151 0.247 0.241 0.412
0.150 0.202 0.277 0.427
3
0.154 0.312 0.267 0.414
10 flasks
Turbidity B
1
2
3
average = average = average = average =
404/ml 587/ml 715/ml 1147/ml
average =
control O.D.= 0.019,1/10
5
average 0.228
Using the equation H = ((Bk - Bn)/(Bk - B0))x100
where
H is percent cell growth inhibition H Bk is the turbidity in the control flasks Bn is the turbidity in the test flasks (after deduction of non-inoculated whites) BO is the turbidity at t = 0 in the control flasks.
We obtain:
Substance concentration (mg/l)
3 2
1 0.5
H%
67.9 %
53%
42.6 % 7.9 %
By drawing the logarithmic Gaussian curve (attached) we obtain :
CE10=g/l CE50=g/l
By calculation (linear regression) we obtain :
CE10= GE50=
0.47 g/l 1.75 g/l
0.466 g/l 1.76 g/l
Additional results:
The pH was measured at the start and end of the test:
At 3 mg/l, at t = 0, pH = 7.03; at t = 16 h 30, pH = 7.10 At 0 mg/l, at t = 0, pH = 6.99; at t = - h, pH = 7.06
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Validity clauses: The test was carried out using the protocol described in document: ISO TC147/SC5/WG1/N111 During the test, cell growth in the control series occurred at a factor of: 12
(BO=10andBk=1299)
The reference substance, 3,5-dichlorophenol gave under the test conditions ; CE 50 = 8.55 mg/l
Ce 10= 0.427 mg/l
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