Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol]and propylidynetrimethanol and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

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:

30/07/12 - 01/08/12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with OECD 209 and GLP standards. As such, this study is considered to be Klimisch 1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

A nominal 5000 mg/l stock solution of HB TMP was prepared in reverse osmosis water which had been heated to 70oC. A nominal 500 mg/l solution was prepared by dilution of the 5000 mg/l stock. Test solutions were prepared by the addition of a known volume of these stock solutions to a total volume of 100 ml reverse osmosis water, synthetic sewage and activated sludge. Nominal concentrations were 10, 100 and 1000 mg/l.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge was obtained from Totnes Sewage Works, Totnes, Devon, UK, on the day of exposure of test and reference substances. This facility treats predominantly domestic sewage. The activated sludge was settled and the supernatent decanted. The sludge was fed with 50 ml of OECD synthetic sewage feed per litre of sludge per day and aerated at room temperature until the test. The total filterable solids concentration was determined to be 7759 mg/l. The pH was 6.9, which was adjusted to 7.2 with 2M sodium hydroxide prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

No information provided

Test temperature:

20± 2oC

pH:

7.4-8.1

Dissolved oxygen:

no information provided

Nominal and measured concentrations:

Nominal concentrations: 10, 100 and 1000 mg/l

Details on test conditions:

See methods below

Reference substance (positive control):

yes

Remarks:

3,5-dichlrophenol

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

Remarks:

respiration rate

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

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

other: EC80

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:

In test flasks dosed with HB TMP, a non-dosed response was observed and < 13% inhibition was observed in all tested concentrations. Respiration rate of the abiotic control was negligible throughout the test. Based on these results the 3-hour EC50 was determined to be > 1000mg/l, while the 3-hour NOEC was determined to be 1000 mg/l, as percentage inhibition less than or equal to 10% was within the expected variability of the test.

Results with reference substance (positive control):

The 3-hour EC50 was found to be 3 mg/l. This was within the expected range for this substance and confirmed the viability of the sludge organisms.

Reported statistics and error estimates:

The percentage inhibition was calculated as:
{1-[respiration rate of test flask/Mean respiration rate of control flasks]} x 100

Table 1: Experimental design

Test substance	Nominal test concentration (mg/l)	Volume of activated sludge (mL)	Volume of synthetic sewage feed (mL)	Volume reference substance stock (mL)	Volume of test substance stock (mL)
Control	Control	19	3.2	-	-
3,5 -DCP	32 (abiotic)	-	3.2	64	-
3,5 -DCP	32	19	3.2	64	-
3,5 -DCP	10	19	3.2	20	-
3,5 -DCP	3.2	19	3.2	6.4	-
3,5 -DCP	1.0	19	3.2	2.0	-
Control	Control	19	3.2	-	-
HB TMP	1000 (abiotic)	-	3.2	-	20
HB TMP	1000	19	3.2	-	20
HB TMP	1000	19	3.2	-	20
HB TMP	100	19	3.2	-	20
HB TMP	10	19	3.2	-	20

Table 2: Results for pH, respiration rate and inhibition.

Test substance	Nominal test concentration (mg/L)	pH		Respiration rate (mg/L/h)	Percentage inhibition
		Start	End
Control	Control	7.5	8.1	31.6	-
3,5 -DCP	32 (abiotic)	7.5	7.4	-	-
3,5 -DCP	32	7.5	8.3	4.8	86
3,5 -DCP	10	7.4	8.4	8.9	74
3,5 -DCP	3.2	7.7	8.4	16.6	51
3,5 -DCP	1.0	7.6	8.3	15.2	55
Control	Control	7.6	8.1	36.1	-
HB TMP	1000 (abiotic)	7.4	7.8	-	-
HB TMP	1000	7.6	8.1	29.9	12
HB TMP	1000	7.5	8.1	35.8	<10
HB TMP	100	7.5	8.0	29.7	12
HB TMP	10	7.6	8.0	33.0	<10

Validity criteria fulfilled:

yes

Conclusions:

Less than 13% inhibition was observed in al HB TMP concentrations. The 3-hour EC50 was estimated to be >1000 mg/L, and the 3-hour NOEC was 1000 mg/L.

Executive summary:

A 3 -hour microbial respiration inhibition test was carried out according to OECD 209 using the test substance HB TMP. A stock solution of the test substance was created and appropriate test solutions were created by addition of activated sludge, synthetic sewage media, and reverse osmosis water. Nominal concentrations were 10, 100 and 1000 mg/L. An abiotic control was also included at 1000 mg/l. Test temperature was 20± 2oC and pH ranged between 7.4 and 8.1. The respiration rate in each flask was determined after 3 hours exposure to the test substance.

Less than 13% inhibition was observed in al HB TMP concentrations. The 3-hour EC50 was estimated to be >1000 mg/L, and the 3-hour NOEC was 1000 mg/L.

Description of key information

The 3-hour EC50 was found to be >1000 mg/L, and the 3-hour NOEC was found to be 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A 3 -hour microbial respiration inhibition test was carried out according to OECD 209 using the test substance. A stock solution of the test substance was created and appropriate test solutions were created by addition of activated sludge, synthetic sewage media, and reverse osmosis water. Nominal concentrations were 10, 100 and 1000 mg/L. An abiotic control was also included at 1000 mg/l. Test temperature was 20± 2oC and pH ranged between 7.4 and 8.1. The respirtaion rate in each flask was determined after 3 hours exposure to the test substance.

Less than 13% inhibition was observed in all concentrations. The 3-hour EC50 was estimated to be >1000 mg/L, and the 3-hour NOEC was 1000 mg/L.