Reaction mass of 2,2'-(vinylenedi-p-phenylene)bis[5-methylbenzoxazole] and 2,2'-(vinylenedi-p- phenylene)bisbenzoxazole and 2-[4-[2-[4-(benzoxazol-2- yl)phenyl]vinyl]phenyl]-5-methylbenzoxazole

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:

August 10, 2017

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:

adopted July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on test solutions:

A stock solution was not prepared; however at the start of the test defined amounts of the test item (3, 30 and 300 mg test item corresponding to the concentrations of 10, 100 and 1000 mg/l; furthermore 3 × 300 mg test item for abiotic controls) were directly weighed (administered) into each test flask and the subsequent calculations refer to the initial weighed nominal concentration. In this study analytical measurements were not performed.
The pH of the test substance-treated mixtures was determined prior to the inoculation to ascertain whether the test item has any effect on the pH.
The pH of the solutions of the item in the test was in the acceptable range (pH 7-8) prior to and after inoculum addition.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary.
- Collection: 09 August 2017 (one day before the test).
- Pretreatment: the coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and the wash-water was removed by centrifuging again. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.
- Preparation of inoculum for exposure: calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to 3 g per litre, based on dry weight.
- Storage: the activated sludge was not used on the day of the collection, but continuously aerated (2 l/minute) at the test temperature for about 24 hours (1 day) and fed with 50 ml synthetic sewage/l activated sludge.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

7.21 - 8.38

Nominal and measured concentrations:

10, 100 and 1000 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer glass bottles of approximately 300 ml volume.
- Aeration: test flasks were incubated under conditions of forces aeration by compressed air (0.5 litre per minute).
- No. of vessels per concentration: 1 replicate at 10 and 100 mg/l; 3 replicates at 1000 mg/l.
- No. of vessels per control: 8 replicates of control group (water, synthetic sewage and inoculum, but without addition of the test or reference item) were tested in parallel, 4 at the start and 4 at the end of the test series.
- No. of vessels per abiotic control: 3 replicates; abiotic controls (water, synthetic sewage and the test item in the corresponding concentration, but without inoculum) were prepared and tested at the highest test item concentration (1000 mg/l) in three parallels.
- No. of vessels per reference control: the reference item 3,5-Dichlorophenol was tested at 3 concentrations, each with 3 parallels.
- No. of vessels per nitrification control: mixtures in 3 parallels (same as the blank controls, however containing 11.6 mg/l N-allylthiourea).
- Sludge concentration: activated sludge concentration in all test mixtures was nominally 1.5 g/l of suspended solids.

TEST MEDIUM / WATER PARAMETERS
- Synthetic sawage: the ratio of components referring to 1000 ml are peptone 16 g, LAB-LEMCO Powder (BEEF extract) 11g, urea 3 g, NaCl 0.7 g, CaCl2 × 2H2O 0.4 g, MgSO4 × 7H2O 0.2 g, K2HPO4 2.8 g, deionized water ad. 1000 ml.
- pH: 7.42 (within the range of 7.5 ± 0.5).
- Storage: prepared synthetic sewage was stored in the dark, in refrigerator at the temperature range of 2.7 - 5.8 °C

EFFECT PARAMETERS MEASURED
The oxygen concentration was measured with O2 electrode (working based on LDO method) under stirred conditions and was recorded for about 6-10 minutes. The measurement was carried out in completely filled Winkler bottles. For practical reason more O2 electrode were used. Simultaneous (a maximum of four vessels were investigated in parallel) measurements were performed; the test vessels were investigated in 8 cycles with the available four O2 electrodes. The oxygen consumption rate (in mg O2 l/h) was determined from the linear part of the respiration curve (in the range between approximately 2 - 7 mg O2/l).

VALIDITY CRITERIA OF THE STUDY
- For 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 coefficient of variation of oxygen uptake rate in control replicates should not be more than 30 % at the end of definitive test.
- The 3-hour EC50 of the reference item 3,5-dichlorophenol (for the used activated sludge batch) should be in the range of 2 mg/l to 25 mg/l for total respiration, 5 mg/l to 40 mg/l for heterotrophic respiration and 0.1 mg/l to 10 mg/l for nitrification respiration.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect (the observed slight 2.85 % stimulation at 10 mg/l, 1.43 % inhibition at 100 mg/l and in average 0.76 % inhibition at 1000 mg/l were within the biological variability of the applied test system) of the test item was observed.
The specific respiration rates of the highest dose, 1000 mg/l were compared with the blank control values using 2 Sample t-Test (α=0.05). No statistical significant differences were observed in the comparison with the blank control values. Based on the results of this study the NOEC was determined to be 1000 mg/l.
Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/l.

ABIOTIC CONTROL
In the experiment abiotic oxygen consumption of the test substance was not observed (in average 1.06 mg/lh); therefore the total oxygen consumption rates were not corrected with the abiotic control values in the subsequent calculations.

NITRIFICATION CONTROL
The measured nitrification potential of the applied activated sludge did not necessitate further testing.
The total respiration (RT) was 44.70 mg/lh, the heterotrophic respiration (RH) was 43.68 mg/lh, the nitrification respiration (RN): 1.02 mg/lh was calculated according to the equation: RN= RT-RH.
The obtained 1.02 mg/lh was considered as not significant difference, being within a biological variability range of the applied test system, that lower than the 5 % of RT (2.23 mg/lh) in blank controls.
According to the above calculations and based on the OECD 209 guideline it was assumed that the heterotrophic oxygen uptake equals the total uptake.

VALIDITY CRITERIA OF THE STUDY
The specific respiration rate of the blank controls (without the test substance or reference substance) was 29.80 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 6.45 %.
The 3-hour EC50 of the reference item 3,5-dichlorophenol (for the used activated sludge batch) was 14.77 mg/l within the range of 2 mg/l to 25 mg/l, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

Results with reference substance (positive control):

In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 1.72 % at the lowest concentration of 2 mg/l and at the nominal concentrations of 7 and 24.5 mg/l, the oxygen consumption rate was inhibited by 27.29 % and 66.71 %, respectively.
The 3-hour EC50 of 3,5-dichlorophenol was calculated to be 14.77 mg/l, (95 % confidence limits: 12.27-18.33 mg/l).

The specific respiration rate (RS) in the test item and control groups

Concentration (mg/l)	Specific Respiration Rate (mg O2/gh)	Average RS (mg O2/gh)
Control	29.94	29.80 CV(%)= 6.45
	29.26
	27.66
	31.94
	31.51
	28.97
	32.07
	27.04
Nitrification control 11.6 mg/l	28.51	29.12 CV(%)= 5.56
	27.89
	30.95
Ref. item 2 mg/l	30.28	29.28 CV(%)= 3.64
	29.42
	28.16
Ref. item 7 mg/l	23.76	21.67 CV(%)= 8.90
	19.96
	21.28
Ref. item 24.5 mg/l	9.12	9.92 CV(%)= 7.27
	10.52
	10.12
Test item 10 mg/l	30.65	30.65
Test item 100 mg/l	29.37	29.37
Test item 1000 mg/l	29.48	29.57^n.s.CV(%)= 0.94
	29.35
	29.89
Abiotic control 1000 mg/l	-0.44	-0.7 CV(%)= -51.71#
	-1.12
	-0.55

CV: Coefficient of variation

n.s.: statistically not significant significantly different compared to the control (2 Sample t-Test; α = 0.05)

#: the extremely high CV value was caused by the low calculated mean values that have a standard deviation of 0.36.

The Q1, Q2 and the applied Δt values in the test item and control groups; the oxygen consumption rate (R), and % inhibition of R

Concentration (mg/l)	Oxygen concentration (mg O2/l)		Δt (min)	Oxygen Consumption Rate (R) (mg O2/lh)	Average R (mg O2/lh)	Inhibition of R (%)
	Q1	Q2
Control	7.26	2.02	7	44.91	44.7	0.00
	7.31	2.19	7	43.89
	7.15	2.31	7	41.49
	7.32	2.13	6.5	47.91
	7.31	2.19	6.5	47.26
	7.22	2.15	7	43.46
	7.15	2.34	6	48.10
	7.28	2.21	7.5	40.56
Nitrification control 11.6 mg/l	7.01	2.02	7	42.77	43.68	2.27
	7.25	2.02	7.5	41.84
	7.11	2.08	6.5	46.43
Ref. item 2 mg/l	7.02	2.1	6.5	45.42	43.93	1.72
	7.14	2.36	6.5	44.12
	7.33	2.05	7.5	42.24
Ref. item 7 mg/l	7.75	1.81	10	35.64	32.5	27.29
	7.48	2.49	10	29.94
	7.19	1.87	10	31.92
Ref. item 24.5 mg/l	7.07	4.79	10	13.68	14.88	66.71
	7.62	4.99	10	15.78
	7.51	4.98	10	15.18
Test item 10 mg/l	7.15	2.17	6.5	45.97	45.97	-2.85
Test item 100 mg/l	7.31	2.17	7	44.06	44.06	1.43
Test item 1000 mg/l	7.14	2.35	6.5	44.22	44.36	0.76
	7.01	2.24	6.5	44.03
	7.36	2.13	7	44.83
Abiotic control 1000 mg/l	8.11	8.21	9	-0.67	-1.06	102.36
	7.87	8.15	10	-1.68
	8.42	8.55	9.5	-0.82

Q1: the oxygen concentration at the beginning of the selected section of the linear phase (mg/l)

Q2: the oxygen concentration at the end of the selected section of the linear phase (mg/l)

Δt: the time interval between these two measurements (min.)

The oxygen concentrations and pH values in the test item groups and controls

Concentration (mg/l)	Oxygen concentration (mg O2/l)		pH
	Start of the 3-hour aeration	End of the 3-hour aeration	Before the addition of inoculum	Start of the 3-hour aeration	End of the 3-hour aeration
Control	7.96	7.64	7.48	7.87	8.36
	7.06	7.31	7.48	7.91	8.35
	6.79	7.49	7.47	7.90	8.35
	7.07	7.72	7.46	7.91	8.28
	7.90	7.71	7.51	7.93	8.37
	7.93	7.58	7.5	7.94	8.37
	7.96	7.55	7.51	7.96	8.35
	8.02	7.64	7.50	7.94	8.34
Nitrification control 11.6 mg/l	7.64	7.36	7.50	7.93	8.33
	7.31	7.25	7.48	7.94	8.37
	7.05	7.11	7.47	7.90	8.27
Ref. item 2 mg/l	7.01	7.39	7.46	7.93	8.26
	7.75	7.14	7.53	7.93	8.22
	7.8	7.68	7.50	7.91	8.19
Ref. item 7 mg/l	7.82	7.75	7.48	7.93	8.31
	7.81	7.48	7.49	7.97	8.23
	7.58	7.19	7.52	7.81	8.32
Ref. item 24.5 mg/l	7.81	7.07	7.53	7.87	8.20
	7.85	7.62	7.52	7.89	8.31
	7.87	7.51	7.51	7.87	8.38
Test item 10 mg/l	8.02	7.15	7.52	7.91	8.31
Test item 100 mg/l	8.04	7.31	7.51	7.92	8.37
Test item 1000 mg/l	7.86	7.51	7.50	7.93	8.36
	8.02	7.01	7.51	7.94	8.32
	8.11	7.36	7.51	7.93	8.36
Abiotic control 1000 mg/l	8.29	8.11	‑ *	7.55	7.27
	8.36	7.87	‑ *	7.54	7.21
	8.36	8.42	‑ *	7.54	7.21

* At the Abiotic controls no inoculum was added; therefore one pH measurement was carried out at the start of the test.

Validity criteria fulfilled:

yes

Conclusions:

EC50 (3h) > 1000 mg/l (nominal)
NOEC (3h) ≥ 1000 mg/l (nominal)

Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions.

The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The preliminary test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption.

The test item was investigate at the nominal concentrations of 10, 100 and 1000 mg/l. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration.

In parallel with the test item treatments 3,5 -dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/l; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated.

Abiotic controls (investigated in three parallels) were prepared containing the test item in the concentration of 1000 mg/l, synthetics sewage feed, but no inoculum. In this test no abiotic oxygen consumption was noticed. The test was performed without pH adjustment.

All validity criteria of the study were met. The average specific respiration rate of the blank was 29.80 mg O₂/g activated sludge (based on dry weight) in an hour with a coefficient of variation of 6.45 %. The 3-hour EC50 of the reference item 3,5 -dichlorophenol was 14.77 mg/l within the range of 2 mg/l to 25 mg/l, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary). The observed oxygen consumption rates consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/l: 29.57 mg O₂/gh), no inhibitory effect of the test item was observed.

Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/l. The NOEC was determined to be 1000 mg/l.

The specific respiration rates at the examined highest test item concentration of 1000 mg/l were compared with the blank control values using 2-Sample T-test (α =0.05). No statistical significant differences were observed in the comparison with the blank control values.

In conclusion, this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/l, thus a definite test was considered unnecessary.

Conclusion

EC50 (3h) > 1000 mg/l (nominal)

NOEC (3h) ≥ 1000 mg/l (nominal)

Description of key information

EC50 (3h) > 1000 mg/l (nominal)

NOEC (3h) ≥ 1000 mg/l (nominal)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The influence of the test item on the activity of the activated sludge was tested according to the method and procedures outlined into the OECD guideline 209. The test item was investigate at the nominal concentrations of 10, 100 and 1000 mg/l. In parallel with the test item treatments 3,5 -dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/l; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. All validity criteria of the study were met. The observed oxygen consumption rates consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/l: 29.57 mg O₂/gh), no inhibitory effect of the test item was observed. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/l. The NOEC was determined to be 1000 mg/l.