Benzonitrile, 4-[(1,6-dihydro-4-hydroxy-6-oxo-2-pyrimidinyl)amino]-

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:

2015-06-11 to 2015-07-03

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

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)
- Method: no stock solution could be prepared due to insufficient water solubility. 1L test bottle were filled with 200 mL of the test item mixtures in Milli-RO water with initial loading rate of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for ~24h. 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rate (3.2, 10, 32, 100, 320 and 1000 mg/L). Optimal contact was ensured applying continuous aeration and stirring.
- Controls: blank controls: test medium without test item and treated in the same way as the test item solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: predominantly domestic sewage treatment plant, "Waterschap Aa en Maas", 's-Hertogenbosch, The Netherlands.
- Preparation of inoculum for exposure: coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ~105°C to determine the amount of suspended solids
- Pretreatment: The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per L of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: 3.0 g/L of sludge
- The pH was 7.8 on the day of testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

not applicable

Test temperature:

19.7-21.7°C

pH:

Before addition of sludge: 7.2-7.6.
After the 3-h exposure period: 7.7-8.3

Dissolved oxygen:

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.

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations:
Combined limit/range-finding test: 10, 100 and 1000 mg/L
Final test: Control, 3.2, 10, 32, 100, 320, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels
- Aeration: 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.
- Number of replicates: 5
- Number of control replicates: 6
- Biomass loading rate: initial loading ca. 3.0 g dw/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: adjusted ISO medium, formulated using RO-water (tap water purified by reverse osmosis) GEON Waterbehandeling, Berkel-Enschot, The Netherlands) 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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): the oxygen consumption was recorded for a period of ~10 min. Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
- 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:

other: ELR50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: no data
- The 3-h ELR10 = 335 mg T002326/L (estimation)

Results with reference substance (positive control):

3-h EC50 = 4.5 mg/L.
This is within the recommended range of 2-25 mg/L confirming suitability of the activated sludge used.

Reported statistics and error estimates:

The 3-h EC50 values were based on Probit Analysis.
The NOEC determination was based on the STUDENT-t test for Homogeneous Variances with a Bonferroni-Holm Adjustment in the final test, alpha=., one sided, smaller).

Validity criteria fulfilled:

yes

Conclusions:

An activated sludge respiration inhibition test (according OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h ELR50 > 1000 mg T002326/L. Therefore it is concluded that 1000 mg/L of the test substance did not have a toxic effect on the respiration rate of activated sludge micro-organisms in this study. The results of the study can be considered reliable without restriction.

Description of key information

In the test flasks dosed with the test substance, <10% respiration inhibition was observed at all the tested concentrations. The 3hour ELR50 for the test substance was determined to be >1000 mg/L. The 3hour NOELR was determined to be 1000 mg/L. Therefore it is concluded that 1000 mg/L of the test substance did not have a toxic effect on the respiration rate of activated sludge micro-organisms in this study.