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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 January 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Non-GLP study, inoculum from industrial source used.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): ethoxyethylamine
- Analytical purity: no data
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge, industrial
Details on inoculum:
- Source: activated sludge from waste water treatment plant treating industrial sewage (sampled on 26 Jan 1987)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
30 min
Test temperature:
ambient (20 ± 2 °C)
Nominal and measured concentrations:
nominal: 761 mg/L (401 mg/L DOC)
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: fill volume: 250 mL
- No. of vessels per concentration (replicates): 1
Reference substance (positive control):
no
Key result
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
> 761 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

No respiration inhibition observed.

Respiration change after 30 min incubation period:

- Respiration rate: 25 mg/L*h (reference value: 25 mg/L*h)

- Respiration inhibition of 0% compared to the mean blank value

Validity criteria fulfilled:
not specified
Endpoint:
toxicity to microorganisms, other
Remarks:
Pseudomonas putida test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 March 1996 - 26 March 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-ethoxyethylamine
- Physical state: liquid
- Analytical purity: >99 %
- Stability under test conditions: stable
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (12500 mg/L) was prepared by stirring the test substance in deionised water for 5 minutes.
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 Braunschweig (German collection of microorganisms in Braunschweig)) 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.
- Pre-treatment: 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 7 h while shaking.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
16 h
Test temperature:
21 ±1 °C
pH:
7.0 -10.8 (see below for details)
Nominal and measured concentrations:
Nominal concentrations: 19.53, 39.06, 78.13, 625, 1250, 5000, 10000 mg/L and 625 mg/L (neutralised), 2500 mg/L (neutralised) and 10000 mg/L (neutralised)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: tubes of glass with flat bottom (nominal volume 50 mL, fill volume: 10 mL) plugged with gas unpermeable teflone caps
- No. of organisms per vessel: cell density = 5 FNU
- No. of vessels per concentration (replicates): 4 inoculated replicates and 1 uninoculated replicate
- No. of vessels per control (replicates): 4 inoculated replicates and 1 uninoculated replicate

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes
- 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.
Reference substance (positive control):
no
Key result
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
82.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks:
(inhibition of cell multiplication)
Remarks on result:
other: non-neutralised test solutions
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
145 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks:
(inhibition of cell multiplication)
Remarks on result:
other: non-neutralised test solutions
Duration:
16 h
Dose descriptor:
other: EC90
Effect conc.:
275 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks:
(inhibition of cell multiplication)
Remarks on result:
other: non-neutralised test solutions
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
750 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks:
(inhibition of cell multiplication)
Remarks on result:
other: neutralised test solutions
Details on results:
The inhibition of bacterial cell multiplication is at least partly due to pH effects. The corresponding neutralised samples showed a significantly lower toxicity compared to the non-neutralised samples.

Optical densities of the bacterial cultures at t = 16 h and pH values of inoculated and uninoculated replicates

Concentration

 (mg/L)

Optical density

(mean, corrected)

Growth

(%control)

Inhibition

(% control)

pH

uninoculated

inoculated

0 h

16 h

(min - max)

Control

0.303

100.0

0.0

7.1

7.2

6.2 - 6.3

19.53

0.302

99.6

0.4

7.4

7.4

6.4 - 6.5

39.06

0.343

113.1

-13.1

7.6

7.7

6.4 - 7.0

78.13

0.284

93.8

6.2

8.8

8.6

6.3 - 6.4

312.5

0.007

2.1

97.9

9.7

9.5

9.4 - 9.5

625

0.007

2.0

98.0

9.9

9.7

9.8 - 9.8

1250

0.007

2.0

98.0

10.1

10.0

10.0 - 10.1

156.25

0.136

44.8

55.2

9.3

9.2

6.2 - 6.4

2500

0.007

1.7

98.3

10.3

10.2

10.2 - 10.3

5000

0.009

2.2

97.8

10.5

10.4

10.5 - 10.5

10000

0.017

2.2

97.8

10.8

10.7

10.7 - 10.8

625*

0.161

52.8

47.2

7.2

7.2

4.3 - 5.0

2500*

0.096

31.4

68.6

7.1

7.1

4.2 - 4.3

10000*

0.072

23.3

76.7

7.0

7.1

4.7 - 4.8

* neutralised samples

Validity criteria fulfilled:
yes
Conclusions:
In this Pseudomonas putida test, the test substance 2-ethoxyethanamine was demonstrated to inhibit cell multiplication with the 16-h EC50 and 16-h EC10 being 145 and 82.5 mg/L, respectively, based on the results obtained in test solutions in which pH was not neutralised. Because of the concentration dependent pH increase caused by the test substance, several neutralised solutions were tested as well, resulting in significantly lower growth inhibition than that observed at equal concentrations in non-neutralised solutions. The resulting 16-h EC50 was 750 mg/L. In conclusion, the pH increasing effect of the test substance was at least partly responsible for the observed effect on cell multiplication.
Endpoint:
toxicity to microorganisms, other
Remarks:
Pseudomonas putida test
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): isopropoxypropylamine
- Analytical purity: not stated
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudomonas putida
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
17 h
pH:
See below for details
Nominal and measured concentrations:
Nominal test concentrations: 15.63, 31.25, 62.5, 125, 250, 500, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Penicillium vessel, closed
- Material, fill volume: glass, 10 mL
- Aeration: no
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 1 mL of a Pseudomonas suspension adjusted to a turbidity of 10 TE/F

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AK medium according to DIN 38412, part 8 (draft)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Photometric determination of light extinction after 17 hours of exposure
Key result
Duration:
17 h
Dose descriptor:
EC10
Effect conc.:
83.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: light extinction
Key result
Duration:
17 h
Dose descriptor:
EC50
Effect conc.:
182 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: light extinction
Duration:
17 h
Dose descriptor:
other: EC90
Effect conc.:
374.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: light extinction

 Conc. (nominal) [mg/L]  Extinction  Difference to Control [%]
 Control 0.287 100 
 15.63 0.339  117.93
 31.25  0.314  109.14
 62.5  0.279  97.04
 125  0.218     75.89
 250  0.055  19.15
 500  0.002  0.78
1000  0.001  0.44

                                  pH values

 Conc. (nominal) [mg/L]  pH after 0 h  pH after 17 h
 Control 7.1 6.5 
 15.63 7.3  6.3
 31.25  7.5  6.3
 62.5  8.3  6.4
 125  9.5     6.6
 250  9.9  7.6
 500  10.2  8.6
1000  10.4  9.1
Conclusions:
In this Pseudomonas putida test, the test substance 3-isopropoxypropylamine was demonstrated to inhibit cell multiplication with the 17-h EC50 and 17-h EC10 being 182 and 83.3 mg/L, respectively, based on the results obtained in test solutions in which pH was not neutralised. Because of the concentration dependent pH increase caused by the test substance, the pH may have been (partly) responsible for the observed effect on cell multiplication.
Endpoint:
toxicity to microorganisms, other
Remarks:
Pseudomonas putida tests and activated sludge respiration inhibition test
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The experimental data of the Pseudomonas putida cell multiplication test with the related substance 2-ethoxyethylamine were used for read across. The experimental data from an activated sludge respiration inhibition test with 2-ethoxyethylamine as well as the experimental data from a Pseudomonas putida cell multiplication test with 3-isopropoxypropylamine, i.e. another related test substance, were included in the read across approach to support the read across justification. The read across justification document is included in IUCLID Section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
82.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks:
inhibition of cell multiplication
Remarks on result:
other: Experimental data on 2-ethoxyethylamine used for read across.
Duration:
17 h
Dose descriptor:
EC10
Effect conc.:
83.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks:
inhibition of cell multiplication
Remarks on result:
other: Experimental data on 3-isopropoxypropylamine used to support the read across strategy.
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
> 761 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Experimental data on 2-ethoxyethylamine used to support the read across strategy.

Description of key information

The key study is a Pseudomona putida cell multiplication study performed with the structurally related substance 2-ethoxyethylamine. The 16-h EC10 value obtained in this study, which was 82.5 mg/L, is used as key value for endpoint coverage and hazard assessment. This value should be considered as a worst case value, as it was obtained based on the observations in test solutions in which pH was not adjusted, whereas the inhibition was significantly lower in test solutions in which pH was adjusted. The pH increase caused by the test substance was at least partly responsible for the observed effects, however, the EC10 value from the non-neutralised solutions is used as a worst case to cover for potential direct intrinsic toxic effects.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
82.5 mg/L

Additional information

No data are available on 3-methoxypropylamine itself. Therefore, data obtained for the related substances 2-ethoxyethylamine and 3-isopropoxypropylamine were included in the dossier. For both related substances, a Pseudomonas putida cell multiplication test is available. The test with 2-ethoxyethylamine yielded a 16-h EC50 and 16-h EC10 of 145 and 82.5 mg/L, respectively, whereas the test with 3-isopropoxypropylamine yielded a 17-h EC50 and 17-h EC10 of 182 and 83.3 mg/L, respectively. In both studies however, a test concentration dependent increase in pH was observed in the test solutions, up to consentrations that are not environmentally relevant but may be harmful to the microorganisms in a sewage treatment plant. Therefore, in the test with 2-ethoxyethylamine, the effect of the test substance was also tested in test solutions in which the pH was adjusted. In these neutralised solutions, a significantly lower growth inhibition was observed compared to that observed at equal concentrations in non-neutralised solutions. The resulting 16-h EC50 was 750 mg/L. Consequently, it can be concluded that the pH increasing effect of the test substance was at least partly responsible for the observed effect on cell multiplication. In the test with 3-isopropoxypropylamine, no neutralised test solutions were tested. However, for this test substance as well, it can be assumed that its pH increasing effect was most likely at least partly responsible for the observed adverse effects in the test organism. As a worst case, and to cover for potentially direct intrinsic toxic effects of the substances, the lowest EC10 value - i.e. the one for 2-ethoxyethylamine - was used for endpoint coverage by read across.

In addition, supporting information from an activated sludge respiration inhibition test with 2-ethoxyethylamine was added to the dossier. This respiration inhibition study (performed according to a method similar to that of OECD guideline 209), performed using industrial sewage sludge from a BASF waste water treatment plant (i.e. possible adaptation of the sludge), reported a 30-min EC10 of > 761 mg/L, based on respiration rate. No data on pH of the test solutions were reported in the study. This information is considered as supporting information in the read across approach.

The read across justification document is attached to IUCLID Section 13.