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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)
GLP compliance:
no
Remarks:
non-animal study which was already available, pre-REACH
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1250 mg/L was prepared in deionised 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:
20 ± 1 °C
pH:
Uninoculated t = 0 h: 6.92 - 10.05
Uninoculated t = 17 h: 6.92 - 8.32
Inoculated t =17 h: 6.71 - 7.78
Nominal and measured concentrations:
Nominal concentrations: control, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 mg/L
HCl-neutralised stock solution was used to test the nominal concentrations 125 and 1000 mg/L in parallel (neutralised to pH 6.92 - 7.78).
Details on test conditions:
TEST SYSTEM
- Test vessel: Penicillin vessels with flattened bottom, nominal volume 50 mL and air permeable silicon-foam caps
- Fill volume: 10 mL
- No. of vessels per concentration (replicates): 4 inoculated replicates
- No. of vessels per control (replicates): 1 uninoculated replicate
- Biomass loading rate: 5 TE/F (optical density)

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

OTHER TEST CONDITIONS
- Photoperiod: continuous light
- Adjustment of pH: no
- Light intensity: 120 µEinstein/m²s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Optical density: With a Zeiss PM 2 DL, 1 cm cuvette at a wavelength of 436 nm. Measurements are made at t = 16 h in all inoculated replicates, whereby the uninoculated replicate of each concentration is used as background measurement.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: control, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 mg/L
- Application method: liquid-dosing from the stock solution
Duration:
16 h
Dose descriptor:
EC10
Effect conc.:
292 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Key result
Duration:
16 h
Dose descriptor:
EC50
Effect conc.:
480 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Duration:
16 h
Dose descriptor:
other: EC90
Effect conc.:
930 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition

Remark

The neutralised sample (1000 mg/L, pH 6.94) caused no toxic effect compared to the not neutralised sample (1000 mg/L, pH 10.05).

Optical densities of the bacteria cultures at t = 16 h

Concentration (mg/L)

E 436 nm

Inoculated

E 436 nm

Uninoculated

Difference

(% of controls)

Control

0.908

---

 

 

3.9

0.949

0.001

0.948

104.46

7.8

0.963

-0.001

0.964

106.14

15.6

0.949

0.000

0.949

104.57

31.3

0.962

-0.001

0.963

106.11

62.5

0.998

-0.002

1.000

110.16

125

0.996

0.003

0.993

109.39

250

0.901

0.003

0.898

98.93

500

0.412

-0.003

0.415

45.69

1000

0.036

-0.002

0.038

4.19

Optical densities of the bacteria cultures exposed to test solutions prepared with a neutralized stock solution at t = 16 h

Concentration (mg/L)

E 436 nm

Inoculated

E 436 nm

Uninoculated

Difference

(% of controls)

Control

0.913

---

 

 

125 neutr.

0.957

0.000

0.957

104.82

1000 neutr.

0.937

0.001

0.936

102.49

 

pH values

Concentration (mg/L)

Uninoculated

0 h

Uninoculated

17 h

Inoculated

17 h

Control

6.92

6.94

7.42

100

10.05

8.32

6.94

500

9.69

7.99

6.71

250

9.41

7.74

7.34

125

8.25

7.46

7.54

62.5

7.36

7.20

7.50

31.3

7.13

7.08

7.49

15.6

7.01

7.01

7.48

7.8

6.96

6.97

7.66

3.9

6.95

6.95

7.77

 

 

 

 

1000 neutr.

6.94

6.92

7.37

125 neutr.

6.92

6.92

7.78

Conclusions:
The 16-hour EC50, based on biomass, was 480 mg/L with the free base.
Executive summary:

The inhibitory effect of N,N-Dimethylbutylamine on cell proliferation of Pseudomonas putida was determined according to an acknowledged national test method, DIN 38412 Part 8. The EC50 (16 h) based on biomass was 480 mg/L with the free base (BASF AG, 1991).

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
(draft, 1984)
GLP compliance:
no
Remarks:
non-animal study which was already available, pre-REACH
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution: 2000 mg test substance in 1000 mL; DOC = 1256 mg/L
Test organisms (species):
activated sludge, industrial
Details on inoculum:
- Laboratory culture: activated sludge from a wastewater plant treating industrial wastewater (BASF AG)
- Concentration of dry substance: 1 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Test temperature:
ambient (20 ± 2 °C)
Nominal and measured concentrations:
nominal: 400 and 800 mg/L
Details on test conditions:
A mean reference value of 41 mg/L*h was used instead of running a blank.
Reference substance (positive control):
no
Duration:
30 min
Dose descriptor:
other: EC20
Effect conc.:
> 800 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

Concentration (mg/L)

Conc DOC-Test substance (mg/L)

Respiration rate (mg O2/L*h)

Change of respiration rate (%)

400

251

45

11

800

502

43

5

 

- blank (mean value): 41 mg O2/L*h

- Increase in respiration rate at 400 and 800 mg/L

- The inhibition of the degradation activity of activated sludge is not anticipated when introduced in appropriate low concentrations
Conclusions:
Respiration of microorganisms contained in activated industrial sludge was not affected at concentrations up to 800 mg/L.
Executive summary:

The inhibitory effect of N,N-Dimethylbutylamine on cell respiration of activated industrial sludge was determined according to an acknowledged national test method, ISO 8192 (Draft, 1984). At concentrations of 400 and 800 mg/L the respiration rate (45 and 43 mg O2/L*h) was slightly increased compared to the blank (mean: 41 mg O2/L*h). Thus, respiration of microorganisms contained in activated industrial sludge was not affected at concentrations up to 800 mg/L (BASF AG, 1986).

Description of key information

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

The EC50 (16 hrs), based on biomass, was 480 mg/L with the free base (BASF AG, 1991).

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Respiration of microorganisms contained in activated industrial sludge was not affected at concentrations up to 800 mg/L (BASF AG, 1986).

Key value for chemical safety assessment

EC50 for microorganisms:
480 mg/L

Additional information

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

In the key study, the inhibitory effect of N,N-Dimethylbutylamine on cell proliferation of Pseudomonas putida was determined according to an acknowledged national test method, DIN 38412 Part 8. The EC50 (16 hrs), based on biomass, was 480 mg/L with the free base (BASF AG, 1991).

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

In the supporting study, the inhibitory effect of N,N-Dimethylbutylamine on cell respiration of activated industrial sludge was determined according to an acknowledged national test method, ISO 8192 (Draft, 1984). At concentrations of 400 and 800 mg/L the respiration rate (45 and 43 mg O2/L*h) was slightly increased compared to the blank (mean: 41 mg O2/L*h). Thus, respiration of microorganisms contained in activated industrial sludge was not affected at concentrations up to 800 mg/L (BASF AG, 1986).