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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Dec 2018 - 01 Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a water-based test solution.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A correction was made for the water content/purity of the test item. All concentrations reported are based on the pure test item.
A stock solution of 10 g/L was prepared by adding test item to 500 mL of tap water purified by reverse osmosis. Magnetic stirring for approximately 15 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution (pH 12.5) corresponding to the target test concentrations were then added to the test media (16 mL synthetic medium and made up to 250 mL with tap water purified by reverse osmosis. Thereafter, 250 mL of activated sludge was added resulting in the required concentrations (see 'Any other information on materials and methods' for details). Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Controls: Test medium without test item, treated in the same way as the test item solutions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The concentration of the suspended solids was determined (3.0 g/L, as used for the test). The initial pH of the sludge was 5.8 on the day of testing. The sludge was buffered to a pH of 7.3 using a 70 g/L sodium bicarbonate solution. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. The mean respiration rate was 21 mg O2/g*h and therefore, within the validity criterion.
- Medium: Adjusted ISO-medium, formulated using tap water purified by reverse osmosis, with the following composition: CaCl2.2H2O: 211.5 mg/L; MgSO4.7H2O: 88.8 mg/L; NaHCO3: 46.7 mg/L; KCl: 4.2 mg/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.2 - 23.0°C. The temperature was outside the range as prescribed by the Guideline. However, as the validity criteria for the controls and reference item were met, this deviation is considered not to have influenced the activity of the sludge.
pH:
At start (t=0 h): 7.4 - 7.5
At end (t=3 h): 7.2 - 7.9
Nominal and measured concentrations:
Nominal: 10, 32, 100, 320, 1000 mg/L (purity corrected test item concentrations)
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass open bottles/vessels, 500 mL fill volume
- Aeration: Clean, oil-free air. The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 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): 8 (only the results of 7 controls were used for calculation of NOEC and EC values due to unreliability of the sensor)
- No. of vessels per reference control (replicates): 1 replicate per concentration (4 concentrations used)
- No. of vessels per abiotic control (replicates): 1 (Combined Limit/Range-Finding Test only, no sludge was added)
- Sludge concentration (weight of dry solids per volume): 1.5 g/L suspended solids in the final test mixture.
- Nitrification inhibitor used: none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Synthetic medium: Synthetic sewage feed, according to OECD 209.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not specified.

EFFECT PARAMETERS MEASURED: Oxygen consumption
- Method: After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls.
- Oxygen recording: determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.
- Temperature: The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Combined Limit/Range-finding study test concentrations: 10 (1 replicate), 100 (1 replicate) and 1000 (triplicate) mg/L. Control (6 replicates) and abiotic control (1 replicate).
- Results used to determine the conditions for the definitive study: yes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Pure test item, corrected for water content
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Pure test item, corrected for water content
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence interval: 170-235 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
481 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Pure test item, corrected for water content
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% confidence interval: 440-526 mg/L
Details on results:
- The combined limit/range-finding showed no inhibition, 39% and 87% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was at a concentration between 100 and 1000 mg/L.
- There was no oxygen uptake from abiotic processes.
- Final Test: The effects observed were slightly lower then what was expected based on the results of the combined limit/range-finding test. No statistically significant inhibition of the respiration rate of the sludge was recorded at 32 mg/L. At higher concentrations the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 10% inhibition at 100 mg/L to 87% at 1000 mg/L. See Table 1 in 'Any other information on results' for details on %inhibition.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (5.6 mg/L in the final test, 95% confidence interval: 4.3 - 6.8 mg/L).
Reported statistics and error estimates:
ECx
- Reference item: 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis
- Test item: probit analysis using linear maximum likelihood regression

NOEC determination: Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller.

Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Final Test – Overview of Results

Treatment

Concentration

(mg/L)

Mean respiration rate

% Inhibition of the respiration rate (mean value)

(mg O2/L h)

(mg O2/g h) (a)

Control

Control

36.73

24.48

 

T1

10

39.13

26.09

-6.54

T2

32

36.09

24.06

1.73

T3

100

33.14

22.10

9.75*

T4

320

27.13

18.09

26.12*

T5

1000

4.82

3.22

86.86*

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

* Statistically significantly different compared to control.

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' section for details on validity criteria.
Conclusions:
The test item was not toxic to waste water bacteria (activated sludge) at or below a concentration of 32 mg/L (NOEC). The EC10 was 200 mg/L (95% confidence interval: 170 - 235 mg/L). The EC50 was 481 mg/L (95% confidence interval: 440 - 526 mg/L).
Executive summary:

In a 3 -h activated sludge respiration inhibition study conducted according to OECD Guideline 209 and according to GLP Principles, microorganisms in activated sludge were exposed to the test item at nominal concentrations of 10, 32, 100, 320 and 1000 mg/L. The test item concentration was corrected for water content, reported concentrations are based on the pure test item.

The test item was not toxic at or below a concentration of 32 mg/L (NOEC), and inhibited respiration up to 87% in the highest test concentration. The EC10 was 200 mg/L (95% confidence interval: 170 - 235 mg/L), the EC50 was 481 mg/L (95% confidence interval: 440 - 526 mg/L). The study met all validity criteria and is considered reliable without restriction.

Description of key information

The test item was not toxic to waste water bacteria (activated sludge) at or below a concentration of 32 mg/L (NOEC, based on the pure test item, corrected for water content). The EC10 was 200 mg/L (95% confidence interval: 170 - 235 mg/L). The EC50 was 481 mg/L (95% confidence interval: 440 - 526 mg/L).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
32 mg/L

Additional information

In a 3 -h activated sludge respiration inhibition study conducted according to OECD Guideline 209 and according to GLP Principles, microorganisms in activated sludge were exposed to the test item at nominal concentrations of 10, 32, 100, 320 and 1000 mg/L. The test item concentration was corrected for water content, reported concentrations are based on the pure test item.

The test item was not toxic at or below a concentration of 32 mg/L (NOEC), and inhibited respiration up to 87% in the highest test concentration. The EC10 was 200 mg/L (95% confidence interval: 170 - 235 mg/L), the EC50 was 481 mg/L (95% confidence interval: 440 - 526 mg/L). The study met all validity criteria and is considered reliable without restriction.