Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-02-12 - 1996-02-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Dimethylpiperazin
- Analytical purity: > 99 %
- Lot/batch No.: Abl.-Nr. 32-1659
Analytical monitoring:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions were prepared by diluting a stock solution (12500 mg/l) with demineralised water.
Test organisms (species):
Pseudomonas putida
Details on inoculum:
- Laboratory culture: The used test strain of Pseudomonas putida DSM 50026 is purchased on regular times from DSM (Deutsche Sammlung von Mikroorganismen in Göttingen (German collection of microorganisms in Göttingen)) and cultured further on slant agar in the Laboratory of Ecotoxicology of BASF AG in Ludwigshafen, Germany.
- Method of cultivation: The stock cultures are kept in slant agar culturing tubes of 50 mL. The inoculation density is one smear from an inoculating loop. The culture is transferred weekly.
- Pretreatment: The pre-cultures are kept in Erlenmeyer flasks with a nominal volume of 250 mL. The vessels are filled with 100 mL pre-culture medium and inoculated with a smear from an inoculation loop with 10 TE/F bacteria suspension at 21 ± 1 °C and incubated for 7h while shaking.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Test temperature:
21 ± 1°C
pH:
Uninoculated t = 0h: 6.9 - 10.1
Uninoculated t = 16h: 7.1 - 9.5
Inoculated t =16h: 6.8 - 9.6
Nominal and measured concentrations:
Nominal concentrations: control, 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000, 10000 mg/L
HCl-neutralised stock solution was used to test the nominal concentrations 156.25, 1250 and 10000 mg/L in parallel (neutralised to pH 6.8 - 7.2).
Details on test conditions:
TEST SYSTEM
- Test vessel: tubes of glass with flat bottom, nominal volume 50 mL, plugged with gas permeable siliconesponge caps
- Fill volume: 10 mL
- No. of vessels per concentration (replicates): 4 inoculated replicates, 1 uninoculated replicate
- No. of vessels per control (replicates): 4 inoculated replicates, 1 uninoculated replicate
- Biomass loading rate: 5 FNU (optical density)

TEST MEDIUM / WATER PARAMETERS
- Test medium: AK-Medium according to DIN 38412, Part 8

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Optical density: at a wavelength of 436 nm. Measurements are made at t=16h in all inoculated replicates, whereby the uninoculated replicate of each concentration is used as background measurement.

Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
651 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Remarks on result:
other: not neutralised
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
1 380 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Remarks on result:
other: not neutralised
Duration:
16 h
Dose descriptor:
other: EC90
Effect conc.:
2 240 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Remarks on result:
other: not neutralised
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
> 1 250 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Remarks on result:
other: neutralised
Details on results:
The inhibition of bacterial cell multiplication at test substance concentrations of >= 1250 mg/L is most likely due to pH effects. In the corresponding neutralised samples no or lower inhibitory effects were observed.

The neutralised samples caused no toxic effect up to a concentration of 1250 mg/L.

Optical densities of the bacteria cultures at t =16h (mean of 4 replicates)

Concentration (mg/L)

Optical density (corrected)

(% of control)

Control

0.349

100

4.88

0.353

101.1

9.77

0.373

107.0

19.53

0.367

105.1

39.06

0.356

102.0

78.13

0.360

103.1

156.25

0.367

105.1

312.5

0.354

101.4

625

0.321

92.0

1250

0.203

58.2

2500

0.003

0.9

5000

0.003

0.9

10000

0.003

0.9

156.25 *

0.371

106.3

1250 *

0.360

103.2

1000*

0.079

22.7

* neutralised

 

pH values

Nominal conc. (mg/l)

pH values

Rep. 1

Rep. 2

Rep. 3

Rep. 4

control

7.2

7.2

7.1

7.2

4.88

7.1

7.2

7.3

7.3

9.77

7.1

7.2

7.3

7.3

19.53

7.2

7.3

7.3

7.2

39.06

7.2

7.3

7.3

7.3

78.13

7.2

7.2

7.3

7.4

156.25

7.3

7.3

7.2

7.3

312.5

7.3

7.3

7.3

7.4

625

7.5

7.5

7.5

7.5

1250

7.8

7.7

7.8

7.7

2500

9.0

9.1

9.1

9.1

5000

9.3

9.3

9.3

9.3

10000

9.5

9.6

9.6

9.6

156.25 *

7.2

7.3

7.2

7.2

1250 *

7.1

7.1

7.0

7.1

1000*

6.8

6.8

6.8

6.8

Validity criteria fulfilled:
yes
Conclusions:
The inhibitory effect of the test substance on the celI multiplication of the bacterium Pseudomonas putida was investigated in a 16-hour static test according to DIN 38412 part 8 (March 1991). The test substance was tested in the concentration range between 4.88 and 10,000 mg/l (nominal).
The reference substance result was within required range, and control variability was valid. The 16-hr EC50 of dimethylpiperazine to activated sludge was found to be > 1000 mg/L. The 16-hr EC10 was 651 mg/L.

Description of key information

The 16-h EC50 of the test substance for the organism Pseudomonas putida was found to be > 1000 mg/L. The EC10 (for the not neutralised sample) was 651 mg/L. The EC10 for the neutralised sample was > 1250 mg/L. The worst-case EC10 of 651 mg/L was used for derivation of the PNEC. An assessment factor of 1 was used as specified in the ECHA guidance (2008) for growth inhibition tests with Pseudomonas putida EC10 values.

Key value for chemical safety assessment

EC50 for microorganisms:
1 380 mg/L
EC10 or NOEC for microorganisms:
651 mg/L

Additional information

A 16-h Pseudomonas putida growth inhibition study was conducted according to DIN 38412 part 8. Concentrations tested were: a control treatment and concentrations between 4.88 and 10,000 mg/L. Above a concentration of about 2000 mg/L the pH-values of the test samples rise over pH 9,9 at the beginning of the test. In these samples a strong inhibition of the bacterial cell multiplication occurs (at 10000 mg/L = 99,1% inhibition, at 1250 mg/L = 41.8% inhibition). The testing of the neutralized samples showed an evidently lower inhibitory effect (at 10000 mg/L neutralized sample = 77.3% inhibition and at 1250 mg/L = 3.2% stimulation). The reference substance result was within the required range, and control variability was within the validity requirements. The 16-h EC50 of 1,4-dimethylpiperazine was found to be 1380 mg/L for the not neutralised sample. The EC10 (for the not neutralised sample) was 651 mg/L. As no other study was available, the results of the Pseudomonas putida growth inhibition study were used for PNEC derivation.