Reaction mass of 4-tert-butyl-2,5-xylenol and 6-tert-butyl-2,4-xylenol

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:

February 17 to April 22, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline that could affect the outcome of the study. One minor deviation is stated.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

(the temperature was in a range of 22-25 ºC instead of 20 ± 2 ºC. This deviations was stated as uncritical, as normal respiration activity of the control could be observed).

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(the temperature was in a range of 22-25 ºC instead of 20 ± 2 ºC. This deviations was stated as uncritical, as normal respiration activity of the control could be observed).

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: The test item was added to the test vessel directly.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Pretreatment: sludge was washed with tap water three times, then centrifuged and re-suspended in tap water.
- Dry Matter:
Pre-treatment: the dry matter was determined as 3.08 g suspended solids/L, giving a concentration of 1.23 g suspended solids/L in the test.
Main test: the dry matter of the activated sludge was determined as 3.78 g suspended solids/L, giving a concentration of 1.51 g suspended solids/L in the test.

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Test temperature:

22-25 ºC

pH:

Main test: 7.8-8.0

Nominal and measured concentrations:

Nominal concentrations: six concentrations ranging from 220 to 4.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Aeration: with purified air
- Type of flow-through (e.g. peristaltic or proportional diluter): approx. 0.75L/min.
- No. of vessels per concentration (replicates):
one replicate/treatment (positive control all experiments, test item treatment 10 and 1 mg/L pre-test)
two replicates/treatment (test item main study and treatment 1000 mg/L pre-test)
- No. of vessels per control (replicates):
two replicates before and two after measuring positive control and test item, respectively

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: < 1.0 mg/L
- Pesticides: < LOD
- Conductivity: 142 µS/cm

TEST CONCENTRATIONS
- Test concentrations:
Pre-test: from 1000 to 1 mg/L (nominal concentration) (4 concentrations)
Main test: from 220 to 4.6 mg/L (nominal concentration) (6 concentrations)

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (CAS 591-35-5)

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: CI not determinable

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 30 - 51 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 71 - 110 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels:
Study 3h-EC50 95% Confidence Interval
Pre-Test 14 mg/L not determinable
Main study 8.2 mg/L 4.2 - 12 mg/L

The test item showed inhibition of the respiration rate of activated sludge in concentrations above 22 mg/L. The highest treatment (220 mg/L) showed a mean inhibition value of 80 %. EC20 and EC50 were derived from the four treatments showing significant inhibition values and good correlation. The pre-test and the main studies show good correlation of the inhibitory effects of the test item. In the main study,O2 consumption in two controls (vessel no. 28 and 36) was considerably higher than in the other nine replicates. These replicate were stated as outliers and were not used for evaluation of the results. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Validity criteria fulfilled:

yes

Conclusions:

The following results for the test item were determined:
 
3h NOEC = 22 mg/L
3h EC20 = 41 mg/L (95% confid. interv.: 30 - 51 mg/l)
3h EC50 = 87 mg/L (95% confid. interv.: 71 - 110 mg/l)

Executive summary:

Two valid experiments were performed. In the pre-test, the test item was tested using four concentrations ranging from 1000 to 1 mg/L (nominal concentration). Duration of the test was three hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 3.08 g suspended solids/L, giving a concentration of 1.23 g suspended solids/L in the test. 3,5-Dichloro-phenole was used as positive control. Four concentrations were tested; an EC50 of 14 mg/L was determined, which lies within the demanded range of 5 – 30 mg/L. As inhibition was observed, a study plan amendment was issued and a main study was performed.

 

The main study was performed using six concentrations, ranging from 220 to 4.6 mg/L (nominal concentration). The dry matter of the activated sludge was determined as 3.78 g suspended solids/L, giving a concentration of 1.51 g suspended solids/L in the test. The EC50 of the positive control was determined as 8.2 mg/L, which lies within the demanded range of 5 – 30 mg/L. The following results for the test item were determined:

3h NOEC = 22 mg/L
3h EC20 = 41 mg/L (95% confid. interv.: 30 - 51 mg/l)

3h EC50 = 87 mg/L (95% confid. interv.: 71 - 110 mg/l)

Description of key information

Key study: experimental result: test according to OECD guideline 209 ane EU method C.11. GLP study.

The 3-h EC50 for the test item was determined to be 87 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

87 mg/L

EC10 or NOEC for microorganisms:

22 mg/L

Additional information

Key study: experimental result: test according to OECD guideline 209 ane EU method C.11.

The main study was performed using six concentrations, ranging from 220 to 4.6 mg/L (nominal concentration).

The following results for the test item were determined:

Effect concentrations

Duration	Endpoint	Effect conc.	Nominal/Measured	Conc. based on	Basis for effect	Remarks (e.g. 95% CL)
3 h	NOEC	22 mg/L		test mat.	respiration rate	CI not determinable
3 h	other: EC20	41 mg/L		test mat.	respiration rate	30 - 51 mg/L
3 h	EC50	87 mg/L		test mat.	respiration rate	71 - 110 mg/L