3,4-dihydro-2H-1,4-benzoxazin-6-ol

Toxicity to microorganisms

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:

From 2020-01-22 to 2020-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Appropriate amounts of test item were weighed into sample flasks and stored overnight in a refrigerator. Before test start the sample flasks were added to the test vessels and test water was added. The composition was stirred intensively.
- Controls: Six controls (pure water, synthetic sewage feed and inoculum, but without addition of the test item) were tested in parallel.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not specified
- Other relevant information:
The reference item 3,5-dichlorophenol was tested at the nominal test concentrations of 1, 4, and 16 mg/L (five replicates for each test concentration) under otherwise identical test conditions. A stock solution of 3,5-dichlorophenol was prepared according to the OECD Guideline No. 209: 0.5 g of 3,5-dichloro-phenol was dissolved in 500 mL pure water. Warm water was used to accelerate the dissolution. The solution was filled up to volume when it had cooled to room temperature. The final pH was 7.3 and therefore in the range of 7 to 8. NaOH was used for the adjustment of the pH.
In parallel a nitrification inhibitor N-allylthiourea (ATU) was tested in the same way with six separate controls and at the identical nominal concentrations of the test item and also the reference item 3,5-dichlorophenol under otherwise identical test conditions.
A stock solution of N-allylthiourea was prepared according to the OECD Guideline No. 209: a stock solution of 2.32 g/L Nallylthiourea was prepared. 2.5 mL of this stock solution were added to an incubation mixture of final volume of 500 mL. This resulted in a final concentration of 11.6 mg ATU/L.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Bensheim, Germany.
- Conditioning/Preparation of inoculum for exposure: The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed with 50 mL synthetic sewage feed per litre and kept aerated at room temperature overnight.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gives a concentration of 1.5 g/L suspended solids in the test medium.
- Initial biomass concentration: see above.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20°C ± 2°C during pre-incubation (3 hours) and evaluation period

pH:

From 7.1 at the start to 5.0-7.6 at the end, in the controls.
From 6.9 (at the start) to 7.8 (at the end) in the test item flasks.

Dissolved oxygen:

From 4.7-7.8 at the start to 6.3-8.0 mgO2/L at the end, in the controls.
From 4.6 (at the start) to 8.7 (at the end) mgO2/L in the test item flasks.

Nominal and measured concentrations:

Nominal 10, 32, 100, 320 and 1000 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 300 mL volume
- Type (delete if not applicable): open / closed : not specified
- Material, size, headspace, fill volume: see above
- Aeration: Compressed air (1.017 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 3 g/L
- Nutrients provided for bacteria: Synthetic Sewage feed:
80 g peptone
55 g meat extract
15 g urea
3.5 g NaCl
2.0 g CaCl2 · 2H2O
1.0 g MgSO4 · 7H2O
14 g K2HPO4
Filled up to 5 litres with deionised water. The pH of this solution was 7.0.
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea
- Biomass loading rate: 1.5 g/L suspended solids in the test medium

TEST MEDIUM / WATER PARAMETERS
For each replicate a test solution with a final volume of 500 mL was tested per treatment in a glass flask. 16 mL synthetic sewage feed and an adequate amount of the test item or an adequate volume of the stock solution of the reference item were filled up with pure water to 250 mL before the start of the test. At the start of the test 250 mL activated sludge inoculum with a sludge concentration of 3.0 g/L suspended solids was added, first to two controls, then to the test solutions of the reference item in increasing concentrations, to further two controls, then to the test item in increasing concentrations and finally to additional two controls. During the 3-hour aeration period the flasks were stirred on a magnetic stirrer to maintain sludge flocs in suspension.

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: not specified
- Light intensity: not specified
- Details on termination of incubation: For the measurement of the respiration rate a well-mixed sample of each test medium was poured into a Karlsruher flask after exactly 3 hours incubation time. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve in the range between 8.8 and 0.8 mg O2/L for total respiration and between 9.9 and 2.3 mg O2/L for heterotrophic respiration. In case of low oxygen consumption the values over a period of 10 minutes were used. In case of a faster drop off below 2 mg O2/L, shorter time intervals were used.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using fewer concentrations than requested by guideline: NA
- Range finding study : no

Reference substance (positive control):

yes

Remarks:

3.5-DCP

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

3.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

393 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

18.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

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 heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

15.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

42.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

21.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not specified
- Effect concentrations exceeding solubility of substance in test medium: NA
- Adsorption (e.g. of test material to the walls of the test container): not specified
- Blank controls oxygen uptake rate: 19.7 mg O2/g*h
- Coefficient of variation of oxygen uptake rate in control replicates: 4.5

Results with reference substance (positive control):

Total Respiration Rate:
The inhibition of the activated sludge treated with the reference item was in the range of 41% to 81% for the test concentrations from 1 until 16 mg/L.
Parameter EC10 EC20 EC50
Value [mg/l] 0.1 0.2 2.1
lower 95%-cl 0.0 0.0 0.2
upper 95%-cl 0.5 1.5 21.0

Heterotrophic Respiration Rate:
The inhibition of the activated sludge treated with the reference item was in the range of 17.3% to 65.3% for the test concentrations from 1 until 16 mg/L.
Parameter EC10 EC20 EC50
Value [mg/l] 0.7 1.6 8.3
lower 95%-cl 0.1 0.3 1.1
upper 95%-cl 3.6 8.0 58.7

Respiration Rate based on Nitrification:
The inhibition of the activated sludge treated with the reference item was in the range of 84% to 109% for the test concentrations from 1 until 16 mg/L.
Parameter EC10 EC20 EC50
Value [mg/l] 0.0 0.1 0.2
lower 95%-cl 0.0 0.0 0.0
upper 95%-cl n.d. n.d. n.d.
n.d.: not determined due to mathematical reasons

Reported statistics and error estimates:

Test item:
ECx Estimation:
Regression Analysis: 3-Parametric normal CDF (cumulative distribution function), non-linear regression without weighting; Optimization method: Levenberg-Marquardt.
3-Parametric normal CDF R2:
Total: 0.705 (A significant amount of variance is explained by the regression model)
Heterotroph: 0.136 (The amount of variance explained by the regression model is not significant.)
Nitrification: 0.481 (A significant amount of variance is explained by the regression model)

NOEC estimation:
Test on Normal Distribution: Shapiro Wilk’s test (α= 0.01)
Variance Homogeneity: Levene’s Test (α= 0.01)
T-test Procedure for treatment comparison and NOEC estimation:
Total: Williams Multiple Sequential t-test (α= 0.05, one-sided smaller)
Heterotroph: Dunnett’s Multiple t-test Procedure (α= 0.05, one-sided smaller)
Nitrification: Williams Multiple Sequential t-test (α= 0.05, one-sided smaller)

Validity Criteria of the Study:

Inoculum Control: The oxygen uptake in the blank controls was 19.7 mg/g/h. The respiration rates of the six controls differed not more than 30%. The value was 22.6% for the total respiration and 25.8% for the heterotrophic respiration. The validity criterion was fulfilled.

Reference Item (Total Respiration): The 3-hour EC50 of the reference item 3,5-dichlorophenol for the total respiration was 2.1 mg/L and therefore in the range of 2 to 25 mg/L for the used activated sludge batch.

Reference Item (Heterotrophic Respiration): The 3-hour EC50 of the reference item 3,5-dichlorophenol for the heterotrophic respiration without nitrification was 8.3 mg/L and therefore also in the range of 5 to 40 mg/L for the used activated sludge batch.

Reference Item (Respiration based on Nitrification): The 3-hour EC50 of the reference item 3,5-dichlorophenol for the oxygen uptake due to nitrification was 0.2 mg/L and therefore in the range of 0.1 to 10 mg/L for the used activated sludge batch.

Validity criteria fulfilled:

yes

Remarks:

See above

Conclusions:

The test item had strong inhibiting effects on the respiration (total respiration and nitrification respiration) and no inhibiting effects on heterotrophic respiration.
The NOEC was determined to be ≥ 1000 mg/L for heterotrophic respiration. No ECx values could be calculated for heterotrophic respiration.
The 3-hour EC10 was found to be 3.7 mg/L (total respiration) and 15.3 mg/L (respiration based on nitrification).
The 3-hour EC50 was found to be 393.0 mg/L (total respiration) and 42.2 mg/L (respiration based on nitrification).

Executive summary:

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions, according to OECD 209 guideline and C.11 method and following GLP. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage feed was measured in the presence of various concentrations of the test item after an incubation period of 3 hours.

The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The result confirms the suitability of the activated sludge used.

The test item  had strong inhibiting effects on the respiration (total respiration and nitrification respiration) and no inhibiting effects on heterotrophic respiration.

The NOEC was determined to be ≥ 1000 mg/L for heterotrophic respiration. No ECx values could be calculated for heterotrophic respiration.

The 3-hour EC10 was found to be 3.7 mg/L (total respiration) and 15.3 mg/L (respiration based on nitrification).

The 3-hour EC50 was found to be 393.0 mg/L (total respiration) and 42.2 mg/L (respiration based on nitrification).

Description of key information

The test item  had strong inhibiting effects on the respiration (total respiration and nitrification respiration) and no inhibiting effects on heterotrophic respiration.

The NOEC was determined to be ≥ 1000 mg/L for heterotrophic respiration. No ECx values could be calculated for heterotrophic respiration.

The 3-hour EC10 was found to be 3.7 mg/L (total respiration) and 15.3 mg/L (respiration based on nitrification).

The 3-hour EC50 was found to be 393.0 mg/L (total respiration) and 42.2 mg/L (respiration based on nitrification).

Key value for chemical safety assessment

EC50 for microorganisms:

42.2 mg/L

EC10 or NOEC for microorganisms:

3.7 mg/L