Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 11 Jun 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 Jul 2010
Deviations:
yes
Remarks:
The final test solutions contained 1.2 g dry matter of sludge/L instead of 1.5 g/L
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Bern, Switzerland (26 Jan 2015)
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Since the test item is well soluble, the test concentrations were prepared by dilution of a stock solution prepared in natural water.
- Controls: 6 replicates containing activated sludge and test medium.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Aeration tank of the municipal biological waste water treatment plant ARA Werdhölzli, Zürich, Switzerland (sampling: 08 Jun 2015, 10:30 AM)
- Preparation of inoculum for exposure: The activated sludge was not directly used after sampling from the treatment plant. To keep the viability of the sludge, 80 mL of synthetic sewage feed was added per Liter activated sludge (the volume taken into account is the volume of settled suspension) and the sludge was aerated overnight. Prior to the tests, the sludge was washed with natural water and then suspended in natural water, at a nominal concentration corresponding to 1.2 g/L dry matter in the final mixture.
- Initial biomass concentration: 1.2 g/L dry matter
- Other: The inoculum was not adapted and not pre-conditioned.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
20.6 - 24.2 °f H
2.06 - 2.42 mmol/L
206 - 242 mg/L
Test temperature:
22 ± 2 °C
pH:
7.0 - 8.0
Conductivity:
441 - 631 µS/cm
Nominal and measured concentrations:
Control, 31.6, 100. 316, and 1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL flasks filled with 500 mL test medium.
- Aeration: Shaking at 150 rpm
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 1.2 g/L
- Nutrients provided for bacteria: Synthetic sewage feed (OECD) prepared with tap water: 16 mL of 100-fold sewage concentrate per 500 mL of final test mixture (to 32 mL/L synthetic sewage in the final mixture).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water. The water quality fullills all legal requirements. Therefore, no chlorination is needed and the water can be used in the tests without any treatment.

OTHER TEST CONDITIONS
- Details on termination of incubation: The oxygen electrode was inserted into a 300 mL BOD bottle and the oxygen concentration recorded for up to 10 min. The O2 consumption rate was transcribed through a y/t potentiometric writer (Recorder REC 101, Pharmacia LKB Biotechnology AB, S-75182 Uppsala) connected to the transformer.
- Other: After addition of the activated sludge suspension, the test vessels were aerated by shaking (150 rpm) for the whole test period of 3 h.
- Other: A sensitivity control (3 concentrations) containing activated sludge, test medium and a reference item (1 replicate per test concentration) was also prepared.

EFFECT PARAMETERS MEASURED:
- Respiration: The oxygen concentration [mg/L] was measured 3 h after addition of the activated sludge suspension (following the starting sequence consisting of the addition of the test/reference items and synthetic sewage) and recorded for up to 10 min with an oxygen electrode (ORION 97-08 equipped with an ORION SA 720 transormer, Hügli-Labortec AG, Abtwil, Switzerland). For the measurements, the electrode was inserted into a 300 mL BOD bottle.
- Dry matter content: The dry matter content of the sludge suspension was determined at the end of the test, by drying the blank controls at 105 °C overnight.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: A non-GLP preliminary test was conducted prior to the definitive test.
- Test concentrations range-finding test: 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, in the non-GLP preliminary test 19% respiration rate inhibition as compared to the blank controls was observed.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
35.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: No
- Blank controls oxygen uptake rate: 25.7 mg O2 per gram of activated sludge per hour (required: > 20 mg oxygen per 1 g of activated sludge dw).
- Coefficient of variation of oxygen uptake rate in control replicates: 8.1% (required: ≤ 30%)
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- Relevant effect levels: EC50 (3 h) = 6.6 mg/L 3, 5-dichlorophenol (required: 2 - 25 mg/L)
Reported statistics and error estimates:
The EC10, EC20, EC50 and EC80 values after 3 h of exposure were calculated with ToxRat Standard Version 2.10 (ToxRat Solutions GmbH, Alsdorf, Germany).
The statistical significance of differences between effects observed for control and test item treatment were determined by Dunnett´s test using the ToxRat Standard Version 2.10 (ToxRat Solutions GmbH, Alsdorf, Germany).

RESPIRATION INHIBITION

The following respiration inhibition of the activated sludge compared to the blank controls was observed: 5% at 31.6 mg/L, 24% at 100 mg/L, 52% at 316 mg/L and 85% at 1000 mg/L.

CALCULATION OF EC10, EC20, EC50 AND EC80

For the calculation of the EC10, EC20 , EC50 and EC80 values the nominal concentrations of the test item in the test media were used, assuming the test item to be stable in the test medium for the duration of the test (3 h).

The calculated EC10, EC20, and EC80 values of the test substance were 35.4 mg/L, 83.5 mg/L and 800 mg/L, respectively. The no observed effect concentration (NOEC) was 31.6 mg/L as determined by Dunnett´s test.

Table 1. Activated sludge respiration rates after 3 h of exposure to the test and reference items and calculation of the inhibition.

 

Nominal concentration

[mg/L]

 

Code

O2 consumption rates

[mg O2 L^-1 h^-1]

 

Respiration inhibition

[%]

 

Respiration inhibition

[%],

mean value

Blank, 0

B1

29.6

6.7

 

 

0.0

B2

29.1

8.1

B3

30.4

4.0

B4

36.0

-13.5

B5

32.9

-3.8

B6

32.2

-1.6

Reference item, 3

R1

22.0

30.5

30.5

Reference item, 10

R2

12.1

62.0

62.0

Reference item, 30

R3

4.8

85.0

85.0

Test item, 31.6

T1-A

33.2

-4.6

 

4.8

T1-B

29.6

6.7

T1-C

27.8

12.2

Test item, 100

T2-A

23.8

25.1

 

24.1

T2-B

25.0

21.2

T2-C

23.4

26.2

Test item, 316

T3-A

14.6

53.9

 

51.8

T3-B

15.2

52.1

T3-C

16.0

49.5

Test item, 1000

T4-A

4.8

84.9

 

84.9

T4-B

4.8

84.9

T4-C

4.8

85.0


Description of key information

EC10 (3 h) = 35.4 mg/L (nominal, OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:
305 mg/L
EC10 or NOEC for microorganisms:
35.4 mg/L

Additional information

There is one key GLP study available investigation the effects of the substance on microorganisms from activated sludge by measuring their respiration rate (carbon and ammonium oxidation) in the presence of the substance, according to the OECD guideline 209.

To investigate the inhibitory effects on total respiration, non-adapted activated sludge from a municipal sewage treatment plant was fed with OECD synthetic sewage and inoculated with nominal concentrations of 31.6, 100, 316, and 1000 mg/L substance for 3 h under aerobic conditions. Blank controls (without test item) and sensitivity controls (with a reference item at three concentration levels) were carried out in parallel. After 3 h, the oxygen consumption rate was measured by inserting an oxygen electrode into a BOD bottle and recording oxygen concentration for up to 10 min.

Respiration inhibition of the activated sludge compared to the blank controls was 5% at 31.6 mg/L, 24% at 100 mg/L, 52% at 316 mg/L and 85% at 1000 mg/L. Effect concentrations were calculated with nominal concentrations. The calculated EC10 and EC20 values were 35.4, and 83.5, respectively. Based on the obtained data, the NOEC (3 h) was 31.6 mg/L.