4-oxo-4-(p-tolyl)butyric acid adduct with 4-ethylmorpholine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms, other

Remarks:

determination of the inhibitory effect of water constituents on bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.08.1995 - 18.08.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: German Water Hazard Classification System (herausgegeben vom Umweltbundesamt, September 1979 LTws - Nr.10)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Test Material:
All dilutions of the test material were freshly prepared in sterile distilled water.
The test material dilution series were prepared from an initial solution containing the initial concentration of the test materialmultiplied by 1.25. (this ensures that the correct concentration of the test material is achieved in each of the test cultures after all the necessary additions).
The dilutions were prepared using 250 ml Erlenmexer flasks sealed with non-absorbant cotton wool such that the dilustions containing 1 part by volume of the initial dilution in 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024,and 2048 parts by volume of distilled water.
the following procedure was employed.
To the first flask in each dilution series 160 ml of the initial solution was added. To each of the remaining flasks 80 ml of distilled water was added.
Starting with the first flask, 80 ml of the initial solution were removed and mixed with the 80 ml of distilled water in the second flask. Next 80 ml of solution were removed from the second flask and mixed with the 80 ml of distilled water in the third flask. This was continued through the remainder of the dilution series, discarding 80 ml of solution from the final flask. Each flask therefore contained 80 ml of the relevant dilution of the test material.
The concentration of the test material was not determined by analysis as it was not a requirement of the Test Guidelines.
Preparation of Test Cultures:
To each of the appropriate number of flasks in the dilution series, the test organism and the relevant stock solutions were added.
Controls:
The controls were prepared at the same time as the test material using 80 ml of sterile distilled water instead of 80 ml of the test material dilution.

Vehicle:

no

Details on test solutions:

Due to limited solubility of the test material the maximum concentration of the initial concentration was 80 mg/l.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

The cultures of the test organism, Pseudomonas putida migula strain designation Berlin 33/2 strain number DMS 50026 used in this study were prepared by inoculating "Nutrient medium for pre-cultures" not more than one day before commencing the test. The growth was harvested from a 1-7 day old stock culture. This bacterial suspension was diluted with further amounts of pre-culture nutrient medium to give a turbidity of about 100 FTU (FTU = Formazine Turbidity Units).
10 ml of this suspension was added to 90 ml of the pre-culture nutrient medium (giving a turbidity of about 10 FTU). After incubation at 25 +/- °C for 5-7 hours, the bacterial suspension had a turbidity of about 50 FTU.

Test type:

other: aerobic

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

16 h

Test temperature:

25 +/- 1 °C

Salinity:

Solution No. 1:
10.0 g NaNO3
2.4 g K2HPO4
1.2 g KH2PO4
1.0 g Yeasts extract

Solution No. 2:
10.0 g NaNO3
2.4 g K2HPO4
1.2 g KH2PO4

Solution No. 3:
44.0 g D(+) Glucose Monohydrate

Solution No. 4:
4.0 g MgSO4 x 7H2O
0.01 g Fe(III) citrate

Nominal and measured concentrations:

The concentrations tested were as follows:
initial concentration = 80 mg/l
1 in 2 = 40 mg/l
1 in 4 = 20 mg/l
1 in 8 = 10 mg/l
1 in 16 = 5 mg/l
1 in 32 = 2.5 mg/l
1 in 64 = 1.25 mg/l
1 in 128 = 0.6 mg/l
1 in 256 = 0.3 mg/l
1 in 512 = 0.15 mg/l
1 in 1024 = 0.078 mg/l
1 in 2048 = 0.039 mg/l

Details on test conditions:

Four dilution series of the test material were prepared from an initial concentration of 80 mg/l determined from the dose range-finding study.
To each of the four flasks, containing 80 ml of the appropriate test material dilution, 2.5 ml of Solution No. 2, 2.5 ml of Solution No. 3 and 5 ml of Solution No. 4 were added.
To three of these flasks, 10 ml of the 50 FTU bacterial suspension was added to give a test culture of about 5 FTU.
To a fourth flask 10 ml of sterile distilled water was added.
This gave triplicate test cultures and an uninoculated test at each test material concentration.
To ten control flasks, 80 ml of sterile distilled water, 2.5 ml of Solution No. 3, 5 ml of Solution No. 4 and 10 ml of the 50 FTU bacterial suspension were added.
After incubation at 25 +/- 1 °C for 16 +/- ^hours the extinction at 436 nm of each of these test and control cultures was determined using a CAMSPEC M330 UV/Vis spectrophotometer. The spectrophotometer was blanked against sterile distilled water for the control cultures and the corresponding uninoculated test for the test cultures.
The following mean extinctions were determined from the spectrophotometric data:
a) the control cultures
b) the three test cultures at each test material concentration
The test results were only deemed valid when the initial turbidity of the control cultures, approximately 5 FTU, had increased by a factor of 100 +/- 10 within the test period. This level of increase in bacterial growth is equivalent to 6 or 7 multiplication stages. A spectrophotometer standardisation graph was used for this purpose.
The extinction values for the test cultures were tabulated and the mean for each loading rate calculated. The control culture values were tabulated separately, and the mean calculated.
The percentage inhibitory effect on cell multiplication was calculated for each test material concentration.
Under the definition of the German Water Hazard Classification Scheme, a substance begins to inhibit cell multiplication at a value 10% below that of the average control culture ie. EC10 This value in mg/l is converted to the negative logarithm to the base ten.

Reference substance (positive control):

no

Key result

Duration:

16 h

Dose descriptor:

IC10

Effect conc.:

> 80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Key result

Duration:

16 h

Dose descriptor:

NOEC

Effect conc.:

>= 80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Validity criteria fulfilled:

yes

Conclusions:

At a concentration of 80 mg/L the test material did not inhibit the test organism, Pseudomonas putida over the stated 10 %.

Executive summary:

A study was performed to determine the acute toxicity of the test material in bacteria (Pseudomonas putida). The method used was designed to meet the requirements of the German Water Hazard Classification "Bewertung wassergefährdender Stoffe" - Herausgegeben vom Umweltbundesamt, September 1979 LTws - Nr. 10; the 'International Organisation for Standardisation' (ISO) procedure for "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonas cell multiplication inhibition test)", and the 'Deutsches Institut für Normung' (DIN) procedure for "Determination of the inhibitory effect of water constituents on bacteria by the Pseudomonas cell multiplication inhibition test".

Following a dose range-finding study, a main study was performed in which three identical dilution series of the test material were inoculated with the test organism Pseudomonas putida to give triplicate test cultures concentrations of 80 mg/l to 0.039 mg/l. The cultures were incubated at 25 +/- 1°C for 16 +/- 1 hours.

It was not possible to produce test cultures at test material concentrations greater than 80 mg/l due to the limited solubility of the test material.

At a concentration of 80 mg/l, the test material did not inhibit the growth of the test organism, over the stated 10%.

Reference 2

Endpoint:

toxicity to microorganisms, other

Remarks:

determination of the inhibitory effect of water constituents on bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15.08.1995 - 18.08.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: German Water Hazard Classification System (herausgegeben vom Umweltbundesamt, September 1979 LTws - Nr.10)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Test Material:
All dilutions of the test material were freshly prepared in sterile distilled water.
The test material dilution series were prepared from an initioal solution containing the initial concentration of the test materialmultiplied by 1.25. (this ensures that the correct concentration of the test material is achieved in each of the test cultures after all the necessary additions).
The dilutions were prepared using 250 ml Erlenmexer flasks sealed with non-absorbant cotton wool such that the dilustions containing 1 part by volume of the initial dilution in 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024,and 2048 parts by volume of distilled water.
the following procedure was employed.
To the first flask in each dilution series 160 ml of the initial solution was added. To each of the remaining flasks 80 ml of distilled water was added.
Starting with the first flask, 80 ml of the initial solution were removed and mixed with the 80 ml of distilled water in the second flask. Next 80 ml of solution were removed from the second flask and mixed with the 80 ml of distilled water in the third flask. This was continued through the remainder of the dilution series, discarding 80 ml of solution from the final flask. Each flask therefore contained 80 ml of the relevant dilution of the test material.
The concentration of the test material was not determined by analysis as it was not a requirement of the Test Guidelines.
Preparation of Test Cultures:
To each of the appropriate number of flasks in the dilution series, the test organism and the relevant stock solutions were added.
Controls:
The controls were prepared at the same time as the test material using 80 ml of sterile distilled water instead of 80 ml of the test material dilution.

Vehicle:

no

Details on test solutions:

Due to limited solubility of the test material the maximum concentration of the initial concentration was 80 mg/l.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

The cultures of the test organism, Pseudomonas putida migula strain designation Berlin 33/2 strain number DMS 50026 used in this study were prepared by inoculating "Nutrient medium for pre-cultures" not more than one day before commencing the test. The growth was harvested from a 1-7 day old stock culture. This bacterial suspension was diluted with further amounts of pre-culture nutrient medium to give a turbidity of about 100 FTU (FTU = Formazine Turbidity Units).
10 ml of this suspension was added to 90 ml of the pre-culture nutrient medium (giving a turbidity of about 10 FTU). After incubation at 25 +/- °C for 5-7 hours, the bacterial suspension had a turbidity of about 50 FTU.

Test type:

other: aerobic

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 h

Test temperature:

25 +/- 1 °C

Salinity:

Solution No. 1:
10.0 g NaNO3
2.4 g K2HPO4
1.2 g KH2PO4
1.0 g Yeasts extract

Solution No. 2:
10.0 g NaNO3
2.4 g K2HPO4
1.2 g KH2PO4

Solution No. 3:
44.0 g D(+) Glucose Monohydrate

Solution No. 4:
4.0 g MgSO4 x 7H2O
0.01 g Fe(III) citrate

Nominal and measured concentrations:

The concentrations tested were as follows:
initial concentration = 80 mg/l
1 in 2 = 40 mg/l
1 in 4 = 20 mg/l
1 in 8 = 10 mg/l
1 in 16 = 5 mg/l
1 in 32 = 2.5 mg/l
1 in 64 = 1.25 mg/l
1 in 128 = 0.6 mg/l
1 in 256 = 0.3 mg/l
1 in 512 = 0.15 mg/l
1 in 1024 = 0.078 mg/l
1 in 2048 = 0.039 mg/l

Details on test conditions:

To each of the duplicate flasks, containing 80 ml of the appropriate test material dilution, 2.5 ml of Solution No. 2, 2.5 ml of Solution No. 3 and 5 ml of Solution No. 4 were added.
To one flask of each series, 10 ml of the 50 FTU bacterial suspension was added to give a test culture of about 5 FTU.
To the other flask of each series, 10 ml sterile distilled water was added.
This gave test cultures and an uninoculated test at each test material concentration.
To the control flask, 80 ml of sterile distilled water, 2.5 ml of Solution No. 2, 2.5 ml of Solution No. 3, 5 ml of Solution No.4 and 10 ml of the 50 FTU bacterial suspension were added.
After incubation at 25 +/- 1 °C for 16 +/- 1 hours the extinction at 436 nm of each of these test and control cultures was determined using CAMSPEC M330 UV/Vis spectrophotometer. The spectrophotometer was blanked against sterile distilled water for control cultures and the corresponding uninoculated dilution for the test cultures.

Reference substance (positive control):

no

Key result

Duration:

16 h

Dose descriptor:

IC10

Effect conc.:

> 80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Key result

Duration:

16 h

Dose descriptor:

NOEC

Effect conc.:

>= 80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Validity criteria fulfilled:

yes

Conclusions:

The results indicated that the test material did not significantly inhibit the growth of the test organism.

Executive summary:

A main study was performed using a test material concentration of 80 mg/l as the initial concentration.

Description of key information

At a concentration of 80 mg/l the test material did not inhibit the test organism, Pseudomonas putida over the stated 10%.