3,4-dihydroxybenzonitrile

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydroxybenzonitrile

EC Number:

241-367-9

EC Name:

3,4-dihydroxybenzonitrile

Cas Number:

17345-61-8

Molecular formula:

C7H5NO2

IUPAC Name:

3,4-dihydroxybenzonitrile

Test material form:

solid: particulate/powder

Remarks:

white to brownish

Details on test material:

Batch 151222

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

/

Test solutions

Vehicle:

no

Details on test solutions:

1.1.1. Test Item – Preparation and Test Concentrations
The batch of CH02906 tested was a white to brownish powder with a purity of 99.9%. No correction was made for the purity/composition of the test item.
A stock solution of 10 g/L was prepared by adding 4.9924 g (combined limit/range-finding test) or 5.0112 g (final test) test item to 1000 mL of Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). Magnetic stirring for 30 minutes followed by sonication for 34 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution (pH 4.4) corresponding to the test concentration were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
Any residual volumes were discarded unless otherwise requested by the Study Director.
1.1.2. Reference Item – Preparation and Test Concentrations
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5-dichlorophenol.
A 3,5-dichlorophenol solution with a final concentration of 1 g/L in Milli-RO water was prepared. The pH as used for the test was 7.7 in both the combined limit/range-finding and final test. The 3,5-dichlorophenol stock solution was stored in a freezer (≤-15℃) until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Four concentrations were tested: 1.0, 3.2, 10 and 32 mg/L.

Test organisms

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. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.9 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Test temperature:

The temperature continuously measured in the temperature control vessels ranged between 19 and 21°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

pH:

The pH in all test vessels, before addition of sludge was between 6.5 and 7.4. After the 3 hour exposure period the pH was between 7.6 and 8.5.

Nominal and measured concentrations:

3.2, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels.
- Aeration: yes. 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): 6
- No. of vessels per vehicle control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 3.0 g/L of sludge

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen uptake rate

TEST CONCENTRATIONS
- Range finding study: Combined Limit/Range-Finding Test
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Effect concentrations 3

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Effect concentrations 4

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Details on results:

The effects observed were in agreement with what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC and ECx values.
No statistically significant inhibition of the respiration rate of the sludge was recorded at 10 mg CH02906 per L. At higher concentrations the inhibitory effect of CH02906 on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 25% inhibition at 32 mg/L to 95% at 1000 mg/L.

The pH in all test vessels, before addition of sludge was between 6.5 and 7.4. After the 3 hour exposure period the pH was between 7.6 and 8.5.
The temperature continuously measured in the temperature control vessels ranged between 19 and 21°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 4.7 (2.0 - 7.5) mg/L

Reported statistics and error estimates:

See table below

Any other information on results incl. tables

Final Test – Overview of the Results

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control	0	58.52	39.01
T1	3.2	57.27	38.18	2.13
T2	10	54.26	36.17	7.28
T3	32	43.98	29.32 *	24.85
T4	100	32.92	21.95 *	43.74
T5	320	9.40	6.26 *	83.94
T6	1000	2.84	1.89*	95.14

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

* Statistically significantly different compared to control.

Effect Parameters

Parameter	CH02906 Concentration (mg/L)
NOEC	10
EC10	12 (5.5 - 19 mg/L)
EC20	29 (17 - 41 mg/L)
EC50	110 (87 – 134 mg/L)

(  ) Between brackets the 95% confidence intervals are given.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of this present test CH02906 was not toxic to waste water bacteria (activated sludge) at or below a concentration of 10 mg/L (NOEC).
The EC10 was 12 mg/L (95% confidence interval: 5.5 - 19 mg/L).
The EC20 was 29 mg/L (95% confidence interval: 17 - 41 mg/L).
The EC50 was 110 mg/L (95% confidence interval: 87 - 134 mg/L).

Executive summary:

The objectiveofthe studywasto evaluate CH02906 for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC₅₀and the no-observed effect concentration (NOEC).

The influence of CH02906 on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based onthe OECD guideline No. 209, 2010.

The batch of CH02906 tested was a white to brownish powder with a purity of 99.9%.

The test started with the preparation of a stock solution of 10 g/L. Magnetic stirring for 30 minutes followed by sonication for 34 minutes was applied to accelerate dissolution and to ensure homogeneity.Volumes of the clear and colourless stock solution corresponding to the test concentration were then added to the test media. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The final test was performed based on the result of a preceding combined limit/range-finding test. Six concentrations were tested, ranging from 3.2 to 1000 mg/L and increasing with a factor 3.2. Five replicates per concentration and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 10 mg CH02906 per L. At higher concentrations the inhibitory effect of CH02906 on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 25% inhibition at 32 mg/L to 95% at 1000 mg/L.

The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

CH02906 was not toxic to waste water (activated sludge) bacteria at a concentration of 10 mg/L.

Text Table 1
The effect parameters for the inhibition of the respiration rate are presented below.

Parameter	CH02906 Concentration (mg/L)
NOEC	10
EC10	12 (5.5 - 19 mg/L)
EC20	29 (17 - 41 mg/L)
EC50	110 (87 – 134 mg/L)

(  ) Between brackets the 95% confidence intervals are given.