Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Mar 2018 - 13 Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
22 Jul 2010
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was pipetted directly into the amber test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis). The test item+Milli-RO water mixtures were magnetically stirred for a short period and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added.
- Differential loading: Yes
- Controls: Test medium without test item, treated in the same way as the test item solutions (6 replicates)

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: No
- Inoculum source: Municipal sewage treatment plant 'Watershap Aa en Maas', 's-Hertogenbosch, The Netherlands
- Preparation of sludge: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added.
- Pretreatment: The batch of sludge was used 1 d after collection. Therefore, 50 mL of synthetic medium ( = sewage feed) was added per Liter of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Sludge concentration (suspended solids): 3.0 g/L, as used for the test
- Final concentration of sludge at test start: 1.5 g/L (250 mL sludge in a final volume of 500 mL test solution)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Test temperature:
20 - 21 °C
pH:
Start: 7.4 - 7.5
End: 8.1 - 8.5
Nominal and measured concentrations:
Control, 100, 180, 320, 560, and 1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles
- Aeration: Yes, continuous aeration with clean, oil-free air throughout the test. The aeration was adjusted such that the dissolved oxygen concentration at the start was > 60-70% saturation (60% air saturation is > 5.0 mg/L at 20 °C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1 (only in combined limit/range-finding test)
- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Nutrients provided for bacteria: Synthetic feed
- Performance of the test: The synthetic medium (= 16 mL sewage feed) and an appropriate amount of test item stock were mixed and made up to 250 mL with Milli-RO water (tap water purified by reverse osmosis). Then, 250 mL activated sludge was added and this was the start of the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO-medium was prepared with RO water (tap water purified by reverse osmosis)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Other: The test vessels were constantly aerated and stirred during exposure.

EFFECT PARAMETERS MEASURED:
- Oxygen consumption: After 3 h contact time, the oxygen consumption was recorded for a period of approximately 10 min. During the measurement, the sample was not aerated but continuously sitrred on a magnetic stirrer.
- Oxygen recording: Oxygen measurements were performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multi-channel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study : Yes, a combined limit/range-finding test was performed, in which the highest concentration level was tested in triplicate and lower concentration levels only in 1 replicate.
- Test concentrations: 10, 100, and 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes. No inhibition, 4% and 32% inhibition of the respiration rate was observed at 10, 100, and 1000 mg/L treatment levels, respectively. The EC50 was above the highest concentration tested (1000 mg/L).
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
446 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence interval: 263 - 561 mg/L
Details on results:
- Adsorption: None
- Blank controls oxygen uptake rate: The mean control oxygen uptake rate was 31 mg oxygen per one gram of activated sludge in the final test and thus was higher than the required 20 mg oxygen/g activated sludge (dw of suspended solids).
- Coefficient of variation of oxygen uptake rate in control replicates: The coefficient of variation of oxygen uptake in control replicates was 8% and thus did not exceed 30%.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 7.1 mg/L, 95% confidence interval: 3.9 - 10.3 mg/L
Reported statistics and error estimates:
Data analysis was performed with ToxRat Professional v3.2.1 (ToxRat Solutions GmbH, Germany).
EC10 and EC20 values were based on Probit analysis using linear maximum likelihood regression, wth the percentage of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.
However, no EC50 values could be calculated for the test item because the effects were < 50% (EC50 > 1000 mg/L).
The NOEC was determined with a Williams Multiple Sequential t-test Procedure (alpha = 0.05, one-sided, smaller). An effect was considered significant if the statistical analysis of the data obtained for the test concentrations showed significant inhibition of the respiration rate compared to the control.

Any other information on results incl. tables

VALIDITY CRITERIA

The study fulfilled the validity criteria of the guideline (Table 1) and is thus valid.

 

Table 1: Validity criteria for OECD 209.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

The mean oxygen uptake of the control was 31 mg oxygen per gram activated sludge.

Yes

The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.

The coefficient of variation of oxygen uptake in control replicates was 8%.

Yes

 

RESULTS

 

No statistically significant inhibition of the respiration rate of the sludge was recorded at a test item concentration of 320 mg/L. At higher concentrations, the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, resulting in 14% inhibition at 560 mg/L and 31% at 1000 mg/L (Table 2).

However, the inhibitory effect on aerobic waste water bacteria observed at 1000 mg/L was below 50%. Therefore, the NOEC was considered to be the highest concentration tested where no statistically significant effects were observed. The effect values are summarized in Table 3.

 

Table 2. Results of the final test.

Treatment

Concentration

[mg/L]

Mean respiration rate

mean % inhibition of the respiration rate

[mg O2/L h]

[mg O2/g h]1

Control

0

46.87

31.25

 

1

100

44.84

29.89

4.35

2

180

46.06

30.70

1.74

3

320

46.13

30.76

1.58

4

560

40.51

27.01*

13.58

5

1000

32.40

21.60*

30.87

1The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

 

Table 3. Effect parameters of final test.

Parameter

test item concentration [mg/L]

NOEC

320

EC10

446 (263 – 561 mg/L)*

EC20

708 (563 – 846 mg/L)*

EC50

> 1000

*The 95% confidence intervals are given in brackets.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.