2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 29, 1995 to July 28, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP and reported with a GLP certificate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None specified

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

STUDY SYSTEM: Inoculum of the aqueous phase of non adapted activated sludge
Reasons for the selection of the study system: Activated sludge from the sewage plant at Hildesheim is well suited as it comprises mostly municipal sewage and hardly industrial chemical waste.
Source: Municipal sewage treatment plant, Hildesheim
Pre-treatment: The activated sludge is maintained in an aerobic condition by aeration for four hours and then homogenized with a mixer.
The sludge is filtered and the filtrate is subsequently used to initiate inoculation.

Duration of test (contact time):

28 h

Initial conc.:

35 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

FUNCTIONAL CONTROL: sodium acetate, puriss. (Fa. Flukal. Batch 317053/1993
Replicates: single
Test concentration: 35 (mg/L)
ThCO2: 1.07 (mg CO2/mg)
TOC (calculated): 10.2 (mg C/L)
Purity: > 99 (%) (acc. safety data sheet of the manufacturer)
Test of identity: aggregate state, consistency and colour of the substance according to the information of the manufacturer. The substance was identified on February 24. 1995.

TEST SUBSTANCE: Lowinox WSP
Replicates: double
Test concentration: 15 (mg/L)
ThCO2: 3.03 (mg CO2/substance)
Molecular formula: C29H40O2
TOC (calculated): 12.4 (mg C/L); 0.827 (mgC/mg)

BLANKS: nutrient solution and inoculum
Replicates: double

TOXICITY CONTROL: not necessary, this was checked with the result: no toxicity in the test concentration (OECD 209)

PROCEDURE
Duration: 28 (d)
Frequency and duration of the application: once per setting up over 28 (d)
Test vessels: 5000 (mL), brown glass
Volume of the test medium: 3000 (mL)
Test medium: mineral nutrient solution acc. to OECD 301 B

PERFORMANCE OF THE TEST
A test of identity was made with the test substance on May 30, 1995.
The theoretical CO2 production (ThCO2) was calculated basing on the molecular formula and a test of bacterial toxicity acc. to OECD 209 was carried out on June 26, 1994 with the concentration 20 (mg/L) (NON-GLP-state).
The following test solutions were prepared in 5 L brown glass bottles as incubation vessels:
- one incubation vessel for the reference compound (R1)
- two incubation vessels for monitoring the activity of the inoculum (K1, K2)
- two incubation vessels for the test compound concentration (P l' P 2)
The necessary amounts of nutrient media and inoculum were placed in each of the incubation vessels. The vessels were then connected to the system for the production of CO2 free air and aerated for 24 h. After 24 (hl the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles.
The test and reference substance concentrations were placed in the incubation vessels, the vessels made up to 3 L with aqua bidest. and connected to the system for the production of CO2 free air.
Incubation took place over a period of 29 days in a temperature range between 22 ± 2 (°C) in a water bath.
On the 28th day 1 mL concentrated HCI was added to each of the vessels. Aeration was continued for a further 24 h and on the 29th day the quantity of CO2 released was determined.

TYPE AND FREQUENCY OF MEASUREMENTS
The temperature of the water bath was measured once every working day.
Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in a basic solution. Back titration of the residual Ba(OH)2 with 0,05 N HCI was carried out three times a week during the first ten days and thereafter twice weekly.
At the beginning and at the end of the test the COD of the test substance solutions was determined.
On the 28th day the pH of all solutions was measured prior to acidification.

Reference substance:

acetic acid, sodium salt

Test performance:

The study was performed acc. to OECD 301 8 and GLP Guidelines.
The quality criteria were full filed according to the guideline:
- The total CO2 production in the blank at the end of the test was less than 40 (mg/L).
- The percentage degradation of the functional control reached the pass level of ≥ 60 (%) before day 14.
- The test temperature remained within the range 22 ± 2 (°C).
- The difference of extremes of replicate values of the test substance at the end of the test was less than 20 (%).

Key result

Parameter:

% degradation (CO2 evolution)

Value:

10 - 12

Sampling time:

28 d

Details on results:

TEST OF IDENTIFICATION
The identity of the test substance was checked. Agreement was ascertained with regard to the state, consistency, colour, melting-point, batch number and the name of the test substance given by the sponsor.

CARBON CONTENT OF THE TEST SUBSTANCE
The ThCO2-production was calculated to be 3.03 (mgCO2/mg substance).
The carbon content was calculated on the basis of the molecular formula and the ThCO2
On this basis the test concentration of 15 (mg/l), with a carbon content of 12.4 (mgC/L) was selected.

TOXICITY TO BACTERIA
There was no bacterial toxicity in the concentration 20 (mg/L).

CO2-PRODUCTION AND BIODEGRADABILITY
The total quantity of CO2 (gross CO2 production) generated by the blank, test and reference samples is the sum of the values of all individual determinations.
For calculation the net CO2 production in test and reference samples the amount of CO2 produced was corrected for endogenous CO2 production in the control groups.
The biological degradation is calculated from the ratio theoretical CO2 production to net CO2 production.
The total amount of CO2 produced in 28 days was analysed by titration in 12 measurements.

RESULTS OF THE CONTROL GROUP
In the control group a maximum quantity of 22.6 (mg CO2/ L) was formed (quality criteria: 0: 40 (mg CO2/L) after 28 days).

RESULTS OF THE FUNCTIONAL CONTROL
The functional control came to a maximum of 100 (%) biodegradabilty after 28 days.
The pass level > 60 [%] was reached by day 6 (quality criteria: degradation > 60 [%] after 14 d).

RESULTS OF THE TEST SUBSTANCE
In the test concentration samples a mean biodegradation rate of 11 (%) was reached by day 28. The difference of extremes of replicate values of the test substance at the end of the test was less than 20 [%].

COURSE AND STAGES OF BIODEGRADATION OF REFERENCE AND TEST COMPOUND
In case of the test substance within 25 days only a degradation rate of 10 (%) was achieved in the test concentration 15 [mg/L].
After 28 days the biodegradability was found to be 10-12 [%].
The pass level of ≥ 60 (%) had not been reached neither in a 10 d-window nor after 28 days. The test substance must be regarded to be not ready biodegradable.
On the 28th day the pH of all solutions was measured prior to acidification (July 27, 1995).
The COD of all solutions was determined at the beginning (June 29, 1995) and at the end of the test (July 28, 1995).
The test temperature was constant at 24 [°C].

Key result

Parameter:

COD

Value:

11 - 28 other: mg/L

Results with reference substance:

In the case of the functional control. the adaptation phase transformed to a degradation phase after 2 days (degradation >10 [%]). The pass level > 60 [%J is reached by day 6.

Total CO₂-production and biodegradability after 28 [d]

CO2-production after 28 [d]		Control mean value	Functional control 35 [mg/L]	Test substance
				15 [mg/L]
Gros	[mg/3 L] [mg/L]	67.8 22.6	191.4 63.8	84.3 28.1	81.3 27.1
Net	[mg/3 L] [mg/L]	-- --	123.6 41.2	16.5 5.5	13.5 4.5
ThCO2	[mg/3 L] [mg/L]	-- --	112.4 37.5	136.4 45.5	136.4 45.5
Biodegr. after 28	[%] [d]	--	100	12	10

 

CO₂-production and biodegradability for all determination points in the control and functional control samples

Test day	Date	Control mv	Functional contr. 35 [mg/L]
		mg CO2	mg CO2		Degr.
			Gros	Net	[%]
1	30.06.	2.5	6.4	3.9	3
4	03.07.	10.2	56.2	46.0	41
6	05.07.	15.2	90.9	75.7	67
8	07.07.	19.9	121.5	101.6	90
11	10.07.	26.2	137.8	111.6	99
15	14.07.	35.3	148.7	113.4	100
18	17.07.	41.6	157.9	116.3	100
21	20.07.	47.2	165.9	118.7	100
25	24.07.	54.6	174.5	119.9	100
28	27.07.	60.7	183.1	122.4	100
29	28.07.	65.8	189.9	124.1	100
29	28.07.	67.8	191.4	123.6	100

mv = mean value

 

CO₂-production and biodegradability for all determination points in the control group and test substance samples

Test day	Date	Control mv	Test substance 15 [mg/L]			Test substance 15 [mg/L]
			mg CO2		Degr.	mg CO2		Degr.
		mg CO2	Gros	Net	[%]	Gros	Net	[%]
1	30.06.	2.5	4.8	2.3	2	3.5	1.0	1
4	03.07.	10.2	15.7	5.5	4	12.7	2.5	2
6	05.07.	15.2	21.6	6.4	5	20.7	5.5	4
8	07.07.	19.9	26.3	6.4	5	27.0	7.1	5
11	10.07.	26.2	34.4	8.2	6	34.0	7.8	6
15	14.07.	35.3	45.2	9.9	7	43.8	8.5	6
18	17.07.	41.6	51.8	10.2	7	50.4	8.8	6
21	20.07.	47.2	58.6	11.4	8	57.7	10.5	8
25	24.07.	54.6	68.8	14.2	10	66.3	11.7	9
28	27.07.	60.7	77.1	16.4	12	74.2	13.5	10
29	28.07.	65.8	82.8	17.0	12	79.5	13.7	10
29	28.07.	67.8	84.3	16.5	12	81.3	13.5	10

Degr. = degradation; mv = mean value

 

Water Parameters, COD

	Blanks		Functional control	Test substance
				15 [mg/L]	15 [mg/L]
pH-value	7.68	7.57	8.00	7.57	7.57
COD [mg/L] Start End	<2 <2	<2 <2	19 <2	11* 25	11* 28

*= the test substance was water insoluble and was directly applicated in the test vessel.

 

Temperature of the water bath on days of titration

Date	30.06.	03.07.	05.07.	07.07.	10.07.	14.07.	17.07.	20.07.	24.07.	27.07.	28.07.
Temp. in water bath [°C]	24	24	24	24	24	24	24	24	24	24	24

 

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The pass level of ≥ 60 (%) had not been reached neither in a 10 d-window nor after 28 days.
The test substance must be regarded to be not ready biodegradable.

Executive summary:

The ready biodegradability was determined in the modified Sturm Test according to OECD Guideline 301 B, EU Method C.4-C and GLP principles from June 29, 1995 to July 28, 1995. The duration was therefore 28 days.

Test substance was the product Lowinox WSP, Batch No. 5E049. No carrier for dissolving the test substance was used.

Test facility was DR.U.NOACK-LABORATORIUM FÜR ANGEWANDTE BIOLOGIE in D-31137 Hildesheim.

The test substance concentration selected as appropriate was 15 (mg/l), corresponding to a carbon content of 12.4 (mgC/L). There was no bacterial toxicity in the test concentration.

The degradation was followed by CO₂analyses, which was produced by the respiration of bacteriae.

The amount of CO₂produced by the test substance is determined for the test period and calculated as the percentage of total CO₂that the test substance could have theoretically produced based on carbon composition. Biodegradability is therefore expressed as a percentage ThCO₂(calculated by molecular formula) and was calculated for each titration of CO₂.

In order to check the activity of the study system sodium acetate was used as a functional control. The functional control reached the pass level > 60 (%) by day 6.

The biological degradation of the test substance is shown graphically in comparison with the readily degradable functional control.

Within 25 days only a mean degradation rate of 10 (%) was achieved in the test concentration 15 [mg/L].

After 28 days the biodegradability was found to be 10-12 (%).

The pass level of ≥ 60 (%) had not been reached neither in a 10 d-window nor after 28 days.

The test substance must be regarded to be not ready biodegradable.

 

Ready Biodegradability of test substance Lowinox WSP

Concentration 15 [mg/L]	Biodegradation [%]
	Test duration [d]
	6	15	21	28
Repl. 1	5	7	8	12
Repl. 2	4	6	8	10

 

Description of key information

Key value determined in laboratory study according to OECD Guideline 301 B and EU Method C.4-C.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The test substance concentration selected as appropriate was 15 (mg/l), corresponding to a carbon content of 12.4 (mgC/L). There was no bacterial toxicity in the test concentration.

The degradation was followed by CO₂analyses, which was produced by the respiration of bacteria.

The amount of CO₂produced by the test substance is determined for the test period and calculated as the percentage of total CO₂that the test substance could have theoretically produced based on carbon composition. Biodegradability is therefore expressed as a percentage ThCO₂(calculated by molecular formula) and was calculated for each titration of CO₂.

In order to check the activity of the study system sodium acetate was used as a functional control. The functional control reached the pass level > 60 (%) by day 6.

The biological degradation of the test substance is shown graphically in comparison with the readily degradable functional control.

Within 25 days only a mean degradation rate of 10 (%) was achieved in the test concentration 15 [mg/L].

After 28 days the biodegradability was found to be 10-12 (%).

The pass level of ≥ 60 (%) had not been reached neither in a 10 d-window nor after 28 days.

The test substance must be regarded to be not ready biodegradable.