Boronic acid, (4-ethylphenyl)-

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:

2006-12-12 to 2007-02-26

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

Version / remarks:

adopted April 04,1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Due to the low solubility of the test item in test water, adequate amounts of the test item were directly weighed into each test flask. A highly homogenous suspension was reached by stirring the test item suspension for about 24 hours at 22 °C in the dark before the experiment was started by adding the activated sludge.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Laboratory culture: Activated sludge, microorganisms from a domestic waste water treatment plant
- Name and location of sewage treatment plant where inoculum was collected: domestic sewage plant Darmstadt, Germany.
- Preparation of inoculum for exposure: The activated sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. The latter procedure was repeated twice. The washed sludge was re-suspended in tap water and sewage feed (50 mL/L) was added. The sludge was kept at room temperature under continuous aeration until use for three days.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

18 - 20 °C

pH:

7.4 – 8.6

Dissolved oxygen:

8.4 – 9.0 mg O2/L

Nominal and measured concentrations:

Nominal conc.: 10, 32, 100, 320 and 1000 mg/L;

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume
- Aeration: With compressed air (approximately 0.8 litre per minute)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per reference control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 3.5 g/L

OTHER TEST CONDITIONS
- Adjustment of pH: No

EFFECT PARAMETERS MEASURED: The oxygen uptake rates of all samples will be determined following an incubation period of 3-h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

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

Remarks on result:

other: A NOEC can be established at 10 mg/L since the respiration Inhibition was less than 20% at this concentration.

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

119.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95 % confidence interval: 65.2 – 220.5 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

27.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95 % confidence interval: 6.5 – 53.5 mg/L

Results with reference substance (positive control):

The 3-hour EC20 of 3,5-Dichlorophenol was calculated to be 3.2 mg/L. Based on the measured data, no 95 % confidence limits could be statistically calculated.

The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 11.3 mg/L. Based on the measured data, no 95 % confidence limits could be statistically calculated.

Toxicity of Test Item

Inhibition of Respiration Rate: In comparison to the inoculum controls the respiration rate of the activated sludge was slightly inhibited by 13.9 % and 24.1 %, at the two lowest nominal concentrations of 10 and 32 mg/L, respectively. At the next higher nominal concentrations of 100 and 320 mg/L, the respiration rate was inhibited by 39.2 % and 72.2 %, respectively, and reached a plateau at the highest nominal test concentration of 1000 mg/L with an inhibition rate of 94.7 %. Test item concentrations exceeding 1000 mg/L were not tested.

Toxicity of 3,5-Dichlorophenol

Inhibition of Respiration Rate: The following nominal concentrations of the positive control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 32, 10 and 3.2 mg/L. In comparison to the controls the respiration rate of the activated sludge was moderately inhibited by 21.5 % at the lowest nominal concentration of 3.2 mg/L. At the nominal concentrations of 10 and 32 mg test item/L, the respiration rate was inhibited by 44.3 and 77.2 %, respectively.

Table 1: Influence of the test item on Oxygen Consumption of Activated Sludge

Flask No.	Treatment	Concentration [mg/L]	Oxygen consumption [mg O2/L min]	Inhibition %	pH-values		Oxygen concentration [mg O2/L]
1	control	—	0.390	—	7.4	8.6	8.5	8.6
10	control	—	0.400	—	7.4	8.6	8.5	8.4
mean			0.395
replicate deviation			2.5
5	Test item	1000	0.021	94.7	7.4	8.5	8.6	8.9
6	Test item	320	0.110	72.2	7.4	8.5	8.5	9.0
7	Test item	100	0.240	39.2	7.4	8.5	8.5	8.8
8	Test item	32	0.300	24.1	7.4	8.5	8.5	8.7
9	Test item	10	0.340	13.9	7.4	8.5	8.5	8.6

 

*: start and end of 3-hour aeration

 

Table 2: Influence of 3,5-Dichlorophenol on Oxygen Consumption of Activated Sludge

 

Flask No.	Treatment	Concentration [mg/L]	Oxygen consumption [mg O2/L min]	Inhibition %	pH-values		Oxygen Concentration [mg O2/L]
					start*	end*	start*	end*
1	control	-	0.390	—	7.4	8.6	8.5	8.6
10	control	-	0.400	—	7.4	8.6	8.5	8.4
mean			0.395
replicate deviation			2.5
2	3,5-DCP 32		0.090	77.2	7.4	8.5	8.6	9.2
3	3,5-DCP 10		0.220	44.3	7.4	8.5	8.5	8.8
4	3,5-DCP 3.2		0.310	21.5	7.4	8.5	8.5	8.7

*: start and end of 3-hour aeration

3,5-DCP: 3,5-dichlorophenol

 

Validity Criteria of the Study

Inoculum Control: The respiration rates of the two controls did not differ by more than 15 % (actual: 2.5 %).

Reference Item: The 3-hour EC₅₀ of the reference item 3,5-Dichlorophenol was determined to be 11.3 mg/L and is thus in the range of 5 to 30 mg/L for the used activated sludge batch.

Dissolved Oxygen: The concentration of dissolved oxygen did not drop below 2.5 mg O₂/L during the incubation period, and just before the measurements of the respiration rates the oxygen concentrations were at least 8.3 mg O₂/L and thus > 6.5 mg O₂/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test according to OECD TG 209, the EC50 of the test item was determined to be 119.3 mg/L (95 % CI: 65.2 – 220.5 mg/L) and the NOEC was established at 10 mg/L.

Executive summary:

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions in a study according to OECD TG 209. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3-hours. The test item was investigated at the nominal concentrations of 10, 32, 100, 320 and 1000 mg/L. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control (at concentrations of 3.2, 10 and 32 mg/L) and two inoculum controls were tested as well. Due to the solubility of the test item, it was weighed directly into the test vessels and stirred for about 24 hours before using in the experiment. The 3-hour EC₅₀ for the positive control 3,5-Dichlorophenol, which was tested in the same way as the test item, was found to be 11.3 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge. In comparison to the inoculum controls the respiration rate of the activated sludge was slightly inhibited by about 14 % at the lowest nominal test concentrations of 10 mg/L. At the next higher concentrations of nominal 32, 100, 320 and 1000 mg/L, a dose related inhibition of the respiration rate was found. At 1000 mg/L, an inhibition of about 95 % was measured. Concentrations exceeding 1000 mg/L nominal were not tested. A NOEC can be established at 10 mg/L since the respiration inhibition was less than 20 % at this concentration. The EC₂₀ of the test item was determined to be 27.9 mg/L (95 % CI: 6.5 – 53.5 mg/L) and EC₅₀ of the test item was determined to be 119.3 mg/L (95 % CI: 65.2 – 220.5 mg/L).

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test according to OECD TG 209, the EC₅₀ of the test item was determined to be 119.3 mg/L (95 % CI: 65.2 – 220.5 mg/L) and the NOEC was established at 10 mg/L (reference 6.1.7-1).

Key value for chemical safety assessment

EC50 for microorganisms:

119.3 mg/L

EC10 or NOEC for microorganisms:

10 mg/L

Additional information

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions in a study according to OECD TG 209. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3-hours. The test item was investigated at the nominal concentrations of 10, 32, 100, 320 and 1000 mg/L. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control (at concentrations of 3.2, 10 and 32 mg/L) and two inoculum controls were tested as well. Due to the solubility of the test item, it was weighed directly into the test vessels and stirred for about 24 hours before using in the experiment. The 3-hour EC₅₀ for the positive control 3,5-Dichlorophenol, which was tested in the same way as the test item, was found to be 11.3 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge. In comparison to the inoculum controls the respiration rate of the activated sludge was slightly inhibited by about 14 % at the lowest nominal test concentrations of 10 mg/L. At the next higher concentrations of nominal 32, 100, 320 and 1000 mg/L, a dose related inhibition of the respiration rate was found. At 1000 mg/L, an inhibition of about 95 % was measured. Concentrations exceeding 1000 mg/L nominal were not tested. A NOEC can be established at 10 mg/L since the respiration inhibition was less than 20 % at this concentration. The EC₂₀ of the test item was determined to be 27.9 mg/L (95 % CI: 6.5 – 53.5 mg/L) and EC₅₀ of the test item was determined to be 119.3 mg/L (95 % CI: 65.2 – 220.5 mg/L).