Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2014-01-15 to 2015-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.9 (Biodegradation: Zahn-Wellens Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Calculated Organic Carbon Content: 308.47 mg C per 1000 mg test item according to non-GLP pre-test.
Storage: At room temperature (20°C ± 5°C), under dry and dark conditions.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Species: Activated sludge, microorganisms from a domestic waste water treatment plant
- Origin: Supplied by the sewage plant Roßdorf, Germany
- Conditioning: The activated sludge used for this study was washed by centrifugation
and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. This procedure was repeated three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre were mixed with test water (see 6.5) and then aerated until use.

Duration of test (contact time):

28 d

Initial conc.:

342 mg/L

Based on:

test mat.

Remarks:

corresponding to about 50 mg DOC/L

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Test System
Species: Activated sludge, microorganisms from a domestic waste water treatment plant
Origin: Supplied by the sewage plant Roßdorf, Germany
Conditioning: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted.
The solid material was re-suspended in tap water and again centrifuged. This procedure was repeated three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined.
Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 4 g dry material per litre were mixed with test water and then aerated until use.

Test Units
Type and Size: Cylindrical glass flasks with 3 litre volume were covered with a plastic lid and aerated using a glass tube.
Identification: Each test unit was uniquely identified with the study number, treatment and replicate number.

Test Conditions
Surrounding Type: Climatic chamber
Temperature: 21 – 22 °C
Aeration: The test flasks were aerated with purified, moistened air.
Light Conditions: Darkness
pH-Value of Test Water: 7.4
pH-Value of Test Solutions: 7.4 – 7.9 (measured at the start of the test)
Test Duration: 28 days

Test Water
Reconstituted Test Water: In deionised water analytical grade salts were added to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2
H2O, 0.5 g NH4Cl filled up with deionised water to 1000
mL volume
b) 22.5 g MgSO4 x 7H2O filled up with deionised water to
1000 mL volume
c) 36.4 g CaCl2 x 2H2O filled up with deionised water to
1000 mL volume
d) 0.25 g FeCl3 x 6H2O filled up with deionised water to
1000 mL volume
In order to avoid preparation of the stock solution d) immediately before use, one drop of concentrated HCl per litre was added.
50 mL of stock solution a) and 5 mL of the stock solutions b)
- d) were combined and filled to a final volume of 5000 mL with deionised water.
The final pH was 7.4.

Course of the Test
Aeration of Test System: The air was led through a bottle containing deionised water to clarify and moisture the air. Also the air was filtered through cotton wool. The air-flow was adjusted to achieve a concentration > 1 mg/L DOC in the test media during the test period.
Sampling: On each sampling day, water evaporation loss was compensated by adding deionized water (except at day 0), deposits were scraped off.
Per sample a volume of approximately 40 mL was taken using a one-way syringe. Samples were taken on day 0 at test start and after 3 hours test duration, and on days 2, 5, 7, 13, 16, 21, 27 and 28.
Sample Preparation: Samples were filtered through a 0.45μm filter (Cellulose mixed ester). The remaining sample was used for analysis.

Reference substance:

diethylene glycol

Preliminary study:

Yes, The DOC of a solution of CHIMEXANE HB was determined prior to test starting in a non-GLP pre-test to be 308.47 mg/L.

Parameter:

% degradation (DOC removal)

Value:

13.4

Sampling time:

28 d

Details on results:

Biodegradation of the Test Item
Percentage Biodegradation: Under the test conditions the mean percentage biodegradation of CHIMEXANE HB reached 13.4 % after 28 days of incubation.
Negative biodegradation values were set zero.
Conclusion: As the biodegradation is below 20%, CHIMEXANE HB is considered to be not biodegradable.

Results with reference substance:

Biodegradation of Reference Item Diethylene glycol
Percentage Biodegradation: The reference item Diethylene glycol was sufficiently degraded to 96.0 % after 13 days, and to 100 % after 28 days of incubation.
Conclusion: The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.

Biodegradation in the Toxicity Control

Percentage Biodegradation: In the toxicity control containing both, the test item and the reference item Diethylene glycol, 40.1% biodegradation was noted within 13 days and 72.8 % biodegradation was determined after 28 days of incubation.

Conclusion: According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge microorganisms because degradation was > 25 % within 14 days.

Measurement of dissolved organic carbon (mg DOC/L) in test flasks during the test period of 28 days

Treatment Group	DOC mg/L
	day 0 (0 h)	day 0 (3 h)	day 2	day 5	day 7	day 13	day 16	day 21	day 27	day 28
control 1	19.27	15.72	16.79	13.08	13.6	17.06	17.23	20.42	13.78	16.2
control 2	15.55	13.34	13.08	14.12	16.07	12.67	18.71	19.04	14.29	15.37
mean	17.41	14.53	14.94	13.6	14.84	14.87	17.97	19.73	14.04	15.78
test item 1	41.91	43.79	39.91	37.68	45.21	54.09	48.05	44.78	38.33	40.67
corrected*	24.5	29.26	24.97	24.08	30.37	39.22	30.08	25.05	24.29	24.89
test item 2	39.76	42.88	39.45	39.18	50.31	52.17	50.71	48.98	40.84	40.78
corrected*	22.35	28.35	24.51	25.58	35.47	37.3	32.74	29.25	26.8	25
Diethylene glycol	66.38	69.95	67.01	47.87	11.04	17.1	17.36		14.44	14.89
corrected*	48.97	55.42	52.07	34.27	0	2.23	0	0	0.4	0
Toxicity control	118.39	113.22	115.64	98.17	100.51	73.96	63.28	55.67	41.53	42.64
corrected*	100.98	98.69	100.7	84.57	85.67	59.1	45.31	35.94	27.49	26.86
test item: CHIMEXANE HB n.a. = measured value below limit of quantification *measured value minus mean value of control; negative values were set zero

Percentage Biodegradation of Test Item and Diethylene glycol during 28 Days of Incubation

Treatment Group	% Degradation*
	day 2	day 5	day 7	day 13	day 16	day 21	day 27	day 28
test item 1	14.66	17.7	0	0	0	14.39	16.99	14.94
test item 2	13.54	9.77	0	0	0	0	5.47	11.82
mean	14.1	13.74	0	0	0	7.2	11.23	13.38
Diethylene glycol	6.04	38.17	100	95.99	100	100	99.28	100
Toxicity control	-2.04	14.31	13.2	40.12	54.09	63.58	72.15	72.79
test item: CHIMEXANE HB n.a.= measured value below limit of quantification *The degradation was calculated using the formula: Dt = (1-((Ct - CB) / (CA - CBA))) * 100 Dt: % degradation at sampling time t Ct: DOC mg/L at sampling time t CB: DOC mg/L of control at sampling time t CA: DOC mg/L at test start (sampling time t=3 h) CBA: mean DOC mg/L of control groups at test start (sampling time t=3 h)

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

As the biodegradation is below 20%, CHIMEXANE HB is considered to be not biodegradable.

Executive summary:

The test item CHIMEXANE HB was investigated for its inherent biodegradability in a Zahn-Wellens/EMPA Test over a period of 28 days. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was determined by following the DOC (Dissolved Organic Carbon) of the test item in the incubation flasks during exposure. As a reference item Diethylene glycol was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

Under the test conditions the mean percentage biodegradation of CHIMEXANE HB reached 13.4 % after 28 days of incubation. As the biodegradation is below 20%, CHIMEXANE HB is considered to be not biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2014-01-21 to 2015-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Not applicable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Calculated Organic Carbon Content: 308.47 mg C per 1000 mg test item according to non-GLP pre-test.
Storage: At room temperature (20°C ± 5°C), under dry and dark conditions.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, industrial (adaptation not specified)

Details on inoculum:

Aerobic activated sludge (microorganisms from industrial waste water treatment plant) was supplied by the Sponsor from an industrial waste water treatment plant from L`Òreal, Belgium.
trifugation
and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. This procedure was repeated three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined.
Based on this ratio, calculated aliquots of washed sludge suspension were mixed with test water corresponding to 4 g dry material per litre.
The dry matter of the sludge suspension was determined before test start and appropriate amounts of the sludge suspension were mixed with test medium to achieve a final concentration of 4 mg dry material per litre.

Duration of test (contact time):

28 d

Initial conc.:

64.8 mg/L

Based on:

test mat.

Remarks:

corresponding to 20.0 mg carbon/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Surrounding Type: Controlled-temperature room
- Temperature: 20°C ± 1°C
- Light Conditions: Darkness
- pH-Value of the test water: 7.6
- Aeration: The activated sludge was aerated with CO2-free air prior to test start.
- Recording: Test conditions were recorded with suitable instruments and documented in the raw data.
- Reconstituted Test Water: Analytical grade salts were added to deionised water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with deionised water to 1000 mL volume. The pH was 7.4.
b) 22.5 g MgSO4 x 7H2O filled up with deionised water to 1000 mL volume
c) 36.4 g CaCl2 x 2H2O filled up with deionised water to 1000 mL volume
d) 0.25 g FeCl3 x 6H2O filled up with deionised water to 1000 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d) immediately before use, one drop of concentrated HCl per litre was added.
For each litre of test medium 10 mL of the stock solution a) and 1 mL of the stock solutions b) - d) were combined and filled up to a final volume of 1000 mL with purified deionised water.

- Preparation of the Test Solutions
Test Item (FT): Appropriate amounts of the test item were weighed into a total volume of test water (2500 mL) and activated sludge (4 g/L suspended solids) was added. The mixture was stirred intensively and appropriate amounts were dispensed into the test vessels and sealed with gas-tight septum caps.
Inoculum Control (FB): Activated sludge (4 g/L suspended solids) was added to the test water (2500 mL). The mixture was stirred intensively and appropriate amounts were dispensed into the test vessels and sealed with gas-tight septum caps.
Procedure Control (FC): Appropriate amounts of the reference item sodium benzoate were weighed into a total volume of test water (1500 mL) and activated sludge (4 g/L suspended solids) was added. The mixture was stirred intensively and appropriate amounts were dispensed into the test vessels and sealed with gas-tight septum caps.
Toxicity Control (FI): The toxicity control contains both, the test item and the reference item. The test item and the reference item were weighed into a total volume of test water (1500 mL) and activated sludge (4 g/L suspended solids) was added. The mixture was stirred intensively and appropriate amounts were dispensed into the test vessels and sealed with gas-tight septum caps.
Sodium Hydroxide Solution 7M: To avoid an additional background carbon entry over the Sodium hydroxide solution, two additional flasks with Sodium hydroxide solution were prepared at each sampling date. The measured inorganic carbon content of the test flasks could not be corrected by this blank value, as misleadingly test water instead of deion water was used for preparation.
Headspace to Liquid Ratio: 1:2
Loading Rates: Test item: 19.9 mg carbon per litre (± 15%) corresponding to approximately 65 mg/L test item.
Reference item: 20.0 mg carbon per litre (± 15%) corresponding to approximately 34 mg/L reference item.
Toxicity control: 20.0 mg carbon per litre (± 15%) corresponding to approximately 32 mg/L test item and 17 mg/L of the reference item.

TEST SYSTEM
Type and Size: Glass bottles with screw-caps and Teflon-coated septa (125 mL nominal, 128 mL total volume)
Identification: Each test unit was uniquely identified with the study number, treatment and replicate number.
Number of Replicates: Three replicates per treatment and per sampling were prepared.
For the day 28 sampling totally 5 replicates per treatment
were prepared.

SAMPLING
Sampling Intervals: FT and FB: days 0, 3, 6, 8, 10, 14, 21, 28
FC and FI: days 0, 6, 14, 28
Test Duration: 28 days

Calculation of Biodegradation: The percentage biodegradation (% D) of the test item and of the reference item sodium benzoate were calculated as:
% D = mg/L CO2 produced (TIC) x 100 / mg/L ThCO2 (test or reference item TIC)
The measured values for CO2-production of the test item/reference item containing treatments were corrected by the control before calculation of biodegradation rates.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

None.

Test performance:

No data.

Parameter:

% degradation (CO2 evolution)

Value:

12.1

Sampling time:

28 d

Remarks on result:

other: mean value (n=5)

Details on results:

Percentage Biodegradation: The criterion for ready biodegradability under the conditions of a CO2 Headspace Test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation. This period should not exceed 10 days. The mean biodegradation after 28 days of CHIMEXANE HB was 12.1% (ThIC); the 10 day window criterion was not passed.
Therefore, CHIMEXANE HB is not considered to be readily biodegradable.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 72% after 14 days and to 76% after 28 days of incubation.The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

In the toxicity control containing both, the test item and reference item, 37% biodegradation was noted within 14 days and 50% biodegradation was determined after 28 days of incubation. According to the guideline the inhibition of the reference item in the toxicity control should be less than 25% at day 28. Considering that the amount of carbon of the reference item is halved in the toxicity control compared to the reference item flasks, the inhibition of sodium benzoate in the toxicity control is below 0%.

According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation in the toxicity control was >25% within 14 days and the inhibition of the reference item was below 25% at day 28.

Inorganic Carbon produced in Test Flasks during the Test Period of 28 Days

Treatment	C-content mg/L	Replicates	Day								Day
			0	3	6	8	10	14	21	28	0	3	6	8	10	14	21	28
			TIC [mg/L]								TIC corrected by the inoculum control [mg/L]
Control		1	1.25	1.88	2.81	2.68	2.75	1.9	4.04	3	-	-	-	-	-	-	-	-
		2	1.34	1.62	3.29	2.11	2.95	2.07	3.07	3.31	-	-	-	-	-	-	-	-
		3	1.77	1.07	3.4	7.08	2.75	2.16	2.7	3.23	-	-	-	-	-	-	-	-
		4	-	-	-	-	-	-	-	3.29	-	-	-	-	-	-	-	-
		5	-	-	-	-	-	-	-	3.22	-	-	-	-	-	-	-	-
		mean	1.45	1.52	3.16	3.96	2.82	2.04	3.27	3.21	-	-	-	-	-	-	-	-
Na- Benzoate	20.012	CV%	19.19	27.3	9.91	68.65	3.99	6.51	21.06	3.85
		1	0.87	-	20.08	-	-	16.25	-	18.48	0	-	16.92	-	-	14.21	-	15.27
		2	1.13	-	18.36	-	-	16.54	-	18.69	0	-	15.2	-	-	14.5	-	15.48
		3	1.03	-	26.28	-	-	16.41	-	18.33	0	-	23.12	-	-	14.37	-	15.12
		4	-	-	-	-	-	-	-	18.3	-	-	-	-	-	-	-	15.09
		5	-	-	-	-	-	-	-	18.3	-	-	-	-	-	-	-	15.09
		mean	1.01	21.57	16.4	18.42	0	18.41	14.36	15.21
		CV%	13.17	19.31	0.89	0.91	n.d.	22.63	1.02	1.1
Chimexane HB	19.988	1	1.32	3.16	3.31	1.83	2.42	2.22	3.23	4.24	0	1.64	0.15	0	0	0.18	0	1.03
		2	1.55	3.05	4.63	3.82	3.58	2.23	4.41	8.53	0.1	1.53	1.47	0	0.76	0.19	1.14	5.32
		3	1.57	3.72	2.19	2.48	2.28	2.09	4.08	3.99	0.12	2.2	0	0	0	0.05	0.81	0.78
		4	-	-	-	-	-	-	-	7.31	-	-	-	-	-	-	-	4.1
		5	-	-	-	-	-	-	-	4.04	-	-	-	-	-	-	-	0.83
		mean	1.48	3.31	3.38	2.71	2.76	2.18	3.91	5.62	0.07	1.79	0.54	0	0.25	0.14	0.65	2.41
		CV%	9.28	10.75	36.15	37.43	25.83	3.62	15.57	38.17	89.42	19.87	150	n.d.	174.74	56.38	90.34	89
Toxicity control	19.906	1	1.18	-	15.77	-	-	9.79	-	14.45	0	-	12.61	-	-	7.75	-	11.24
		2	1.43	-	14.04	-	-	8.88	-	13.79	0	-	10.88	-	-	6.84	-	10.58
		3	1.2	-	14.43	-	-	9.4	-	11.89	0	-	11.27	-	-	7.36	-	8.68
		4	-	-	-	-	-	-	-	13.37	-	-	-	-	-	-	-	10.16
		5	-	-	-	-	-	-	-	12.3	-	-	-	-	-	-	-	9.09
		mean	1.27		14.75			9.36		13.16	0		11.59			7.32		9.95
		CV%	11.08		6.15			4.89		8.03	n.d.		7.83			6.25		10.62

Biodegradation of Test Item, Reference Item and Toxicity Control during the Test Period of 28 Days

Treatment	C-content mg/L	Replicates	Day
			0	3	6	8	10	14	21	28
			% biodegradation [net measured TIC/ initial C x 100]
Na- Benzoate	20.012	1	0	---	84.5	---	---	71	---	76.3
		2	0	---	76	---	---	72.4	---	77.3
		3	0	---	115.5	---	---	71.8	---	75.5
		4	---	---	---	---	---	---	---	75.4
		5	---	---	---	---	---	---	---	75.4
		mean	0	---	92	---	---	71.7	---	76
Chimexane HB	19.988	1	0	8.2	0.8	0	0	0.9	0	5.2
		2	0.5	7.7	7.4	0	3.8	0.9	5.7	26.6
		3	0.6	11	0	0	0	0.2	4.1	3.9
		4	---	---	---	---	---	---	---	20.5
		5	---	---	---	---	---	---	---	4.1
		mean	0.4	9	2.7	0	1.3	0.7	3.3	12.1
Toxicity control	19.906	1	0	---	63.3	---	---	38.9	---	56.5
		2	0	---	54.7	---	---	34.4	---	53.1
		3	0	---	56.6	---	---	37	---	43.6
		4	---	---	---	---	---	---	---	51.1
		5	---	---	---	---	---	---	---	45.7
		mean	0	---	58.2	---	---	36.8	---	50

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The degradation rate of CHIMEXANE HB did not reach 60% within the 10-day window and after 28 days.
Therefore, CHIMEXANE HB is considered to be not readily biodegradable.

Executive summary:

The biodegradation of Chimexane HB was studied using non adapted activated sludge from an industrial plant according to OECD 310 with GLP statement.

The criterion for ready biodegradability under the conditions of a CO2 Headspace Test is the 10-day window, describing the period between reaching at least 10% degradation and 60% degradation.

This period should not exceed 10 days. The mean biodegradation after 28 days of CHIMEXANE HB was 12.1% (ThIC); the 10 day window criterion was not passed.

Therefore, CHIMEXANE HB is not considered to be readily biodegradable.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2009-06-04 to 2010-02-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was performed according to the OECD 301B Guideline and the EU Method C.4-C with GLP statement. This study was well documented but doesn't meet all validity criteria. The high variability between replicates was observed at both concentrations, leading to the fact that the validity criterion related to the difference between replicates at the end of the test (or at the plateau) was not fulfilled.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

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

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Non adapted activated sludge from Hildesheim plant which receives predominantly municipal sewage.
Pre-treatment: the sludge was washed twice with autoclave tap water, the settled sludge was filled up with mineral salts medium and maintained in aerobic condition by aerating for 4h. After homogenisation, the supernant was decanted and maintained in aerobic condition by aeration with CO2 free air for 6 days. 10 mL/L were used to initiate inoculation.
Colony forming units in the test vessel: 10E7 - 10E8 CFU/L.

Duration of test (contact time):

28 d

Initial conc.:

40 mg/L

Initial conc.:

11.6 mg/L

Based on:

DOC

Initial conc.:

18 mg/L

Initial conc.:

5.1 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Two independent studies were carried out.

TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 301B / CO2 Evolution test.
- Test temperature: 20.0-23.5°C (1st study); 20.0-23.0°C (2nd study)
- Aeration: 30-100 mL/min
- Photoperiod: Low light conditions
- Dispersion treatment: Continuous stirring

TEST SYSTEM
- Culturing apparatus: Test vessels 5000 mL brown glass, volume of the test medium 3000 mL.
- Number of culture flasks/concentration: 2
- Measuring equipment: Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in an alkaline solution (0.0125 mol/L Ba(OH)2). For each titration the first gas wash bottle was removed and a new bottle was connected to the last one.
Backtitration of the residual Ba(OH)2 with 0.05 N HCl was carried out three times a week during the first ten days and thereafter twice weekly.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes in duplicates (test medium without test and reference item)
- Abiotic sterile control: No
- Toxicity control: Yes in duplicates (test item and reference item in test concentration)

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The first study was conducted with the test item (40 mg/L in duplicates, corresponding to a carbon content (TOC) of 11.6 mg/L). The degradation kinetic of the test item was different between the two test item replicates. In addition, the course of the degradation in the toxicity control indicated a possible inhibitory effect of the test item on activated sludge activity. So the study was repeated using a lower test item concentration (18mg/L).

Test performance:

1st study: ThCO2 = 1.07 mg CO2/mg test item.
2nd study: ThCO2 = 1.04 mg Co2/mg test item.

Parameter:

% degradation (CO2 evolution)

Value:

29

Sampling time:

28 d

Remarks on result:

other: 1st study

Parameter:

% degradation (CO2 evolution)

Value:

42

Sampling time:

28 d

Remarks on result:

other: 2nd study

Details on results:

- Toxicity control: A biodegradation rate of 32% was determined within 14 days in the 1st study and it came to 51% after 28 days. The course of the degradation in the toxicity control indicated a possible inhibitory effect of the test item on activated sludge activity. In the 2nd study, a biodegradation rate of 49% was determined within 14 days and it came to 68% after 28 days.
- Test item: In the 1st study, the 1st test item replicate reached the 10% level after 15 days and came to a biodegradation rate of 43% after 28 days. The 2nd test item replicate reached the 10% level after a long lasting adaptation phase of 28 days with a biodegradation rate of 14%. The mean biodegradation rate after 28 days was 29%. In the 2nd study, the 1st test item replicate reached the 10% level after a long lasting adaptation phase of 25 days and came to a biodegradation rate of 30% after 28 days. The 2nd test item replicate reached the 10% level after an adaptation phase of 12 days and came to a biodegradation rate of 54% after 28 days. The mean biodegradation rate after 28 days was 42%.
See tables in "Any other information on results incl. tables".

Results with reference substance:

The course of the degradation was rapid. In the 1st study, the functional control reached the pass level of 60% after 7 days and came to a maximum of 77% after 28 days. In the 2nd study, the functional control reached the pass level of 60% after 6 days and came to a maximum of 100% after 17 days. The validity criterion degradation >= 60% after 14d for the reference substance is fulfilled in both studies.

Table 5.2.1/1: 1st study: biodegradation of Chimexane HB in comparison to the functional control and toxicity control

	Biodegradation [%]
	Study day[d]
	6	14	21	28
Test item, 1st replicate 40 mg/L	2	9	32	43
Test item, 2nd replicate 40 mg/L	0	0	3	14
Functional control 20 mg/L	57	76	76	77
Toxicity control 40 mg/L test item + 20 mg/L reference item	23	32	39	51

Table 5.2.1/2: 2nd study: biodegradation of Chimexane HB in comparison to the functional control and toxicity control

	Biodegradation [%]
	Study day[d]
	7	14	21	28
Rest item, 1st replicate 18 mg/L	0	0	0	30
Test item, 2nd replicate 18 mg/L	2	17	24	54
Functional control 20 mg/L	69	95	100	100
Toxicity control 18 mg/L test item + 20 mg/L reference item	37	49	58	68

Validity criteria fulfilled:

no

Remarks:

The high variability between replicates was observed at both concentrations

Interpretation of results:

other: not readily biodegradable

Conclusions:

The biodegradation of Chimexane HB was < 60% after 28 days in the CO2 evolution (modified Sturm) test (OECD 301 B).
Under the test conditions, Chimexane HB must be regarded as not readily biodegradable.

Executive summary:

The biodegradation of Chimexane HB was studied using non adapted activated sludge from a predominantly municipal plant according to OECD 301 B and EU method C.4-C with GLP statement at Dr.U.Noack-Laboratorien.

Two independent studies were carried out; on the first study the test item concentration was 40 mg/L in duplicates, corresponding to a carbon content (TOC) of 11.6 mg/L. the degradation kinetic and the course of degradation indicate a possible inhibitory effect of the test item on the activated sludge activity. Therefore the study was conducted using a lower concentration of test item (18 mg/L).

In the first study, the first test item replicate reached the 10% level after 15 days and came to a biodegradation rate of 43% after 28 days. The second test item replicate reached the 10% level after a long lasting adaptation phase of 28 days with a biodegradation rate of 14%. The mean biodegradation rate after 28 days was 29%.

In the second study, the first test item replicate reached the 10% level after a long lasting adaptation phase of 25 days and came to a biodegradation rate of 30% after 28 days. The second test item replicate reached the 10% level after an adaptation phase of 12 days and came to a biodegradation rate of 54% after 28 days. The mean biodegradation rate after 28 days was 42%.

The high variability between replicates was observed at both concentrations, leading to the fact that the validity criterion related to the difference between replicates at the end of the test (or at the plateau) was not fulfilled.

The biodegradation of Chimexane HB was < 60% after 28 days in the CO2 evolution (modified Sturm) test. Under the test conditions, Chimexane HB must be regarded as not readily biodegradable.

Description of key information

Biodegradation of the test item has been investigated during two Ready Biodegradability studies, and during one Inherent Biodegradability syudy. The test item CHIMEXANE HB has been found to be not ready biodegradable and not inherently biodegradable under test conditions.

- OECD Guideline 301B, EU Method C.4-C, GLP, supporting study, validity 2:

First study: 29% biodegradation after 28 days (14-43%).

Second study: 42% biodegradation after 28 days (30-54%).

Non readily biodegradable

- OECD Guideline 310, ISO 14593, GLP, key study, validity 1:

 12.1% biodegradation after 28 days.

Non readily biodegradable

- OECD Guideline 302B, EU Method C.9, GLP, supporting study, validity 1:

13.4% biodegradation after 28 days.

Non inherently biodegradable

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Three studies are available to assess the biodegradation of Chimexane HB.

One OECD 301 B study was performed using non adapted activated sludge from a predominantly municipal plant with GLP statement.

Two independent studies were carried out; on the first study the test item concentration was 40 mg/L in duplicates, corresponding to a carbon content (TOC) of 11.6 mg/L. the degradation kinetic and the course of degradation indicate a possible inhibitory effect of the test item on the activated sludge activity. Therefore the study was conducted using a lower concentration of test item (18 mg/L).

In the first study, the first test item replicate reached the 10% level after 15 days and came to a biodegradation rate of 43% after 28 days. The second test item replicate reached the 10% level after a long lasting adaptation phase of 28 days with a biodegradation rate of 14%. The mean biodegradation rate after 28 days was 29%. In the second study, the first test item replicate reached the 10% level after a long lasting adaptation phase of 25 days and came to a biodegradation rate of 30% after 28 days. The second test item replicate reached the 10% level after an adaptation phase of 12 days and came to a biodegradation rate of 54% after 28 days. The mean biodegradation rate after 28 days was 42%.

The high variability between replicates was observed at both concentrations, leading to the fact that the validity criterion related to the difference between replicates at the end of the test (or at the plateau) was not fulfilled.

The biodegradation of Chimexane HB was < 60% after 28 days in the CO2 evolution (modified Sturm) test. Under the test conditions, Chimexane HB must be regarded as not readily biodegradable.

One OECD 310 study was performed using non adapted industrial sludge with GLP statement.

During this study, the mean biodegradation after 28 days of CHIMEXANE HB was 12.1% (ThIC); the 10 day window criterion was not passed.

Therefore, CHIMEXANE HB is considered to be not readily biodegradable.

One OECD 302B study was performed using activated sludge from the aeration tank of a domestic waste water treatment plant, GLP statement.

Under the test conditions the mean percentage biodegradation of CHIMEXANE HB reached 13.4 % after 28 days of incubation. As the biodegradation is below 20%, CHIMEXANE HB is considered to be not biodegradable.