6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum: Aereation stage of the HRC Limited sewage treatment plant treating predominantly domestic sewage.
- Preparation of inoculum for exposure: The sample was allowed to settle and the supernatant filtered through Whatman GFA filtrer paper (first 250 mL discarded).

Duration of test (contact time):

28 d

Initial conc.:

40 mg/L

Based on:

test mat.

Initial conc.:

31.5 mg/L

Based on:

IC (inorganic carbon)

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Standard nutrient medium
Solution 1:
KH2PO4 8.5 g/L
K2HPO4 21.75 g/L
Na2HPO4 . H2O 33.3 g/L
NH4Cl 1.7 g/L

Solution2:
MgSO4 . 7 H2O 22.5 g/L

Solution 3:
CaCl2 27.5 g/L

Solution 4:
FeCl3 . 6 H2O 0.25 g/L

1 mL of each solution 1 – 4 were added to each litre of aerated reverse osmosis purified and glass-distilled water. The “dilution water” was left at room temperature (ca. 20 °C), under gentle agitation for 24 h prior to use.

- Test temperature: 21 ± 1 °C

TEST SYSTEM
- Culturing apparatus: BOD bottles (280 mL, darkened glass)
- Number of culture flasks/concentration: 2
- Measuring equipment: Yellow Springs BOD Probe (model 54)
- Test performed in closed vessels

SAMPLING
- Sampling frequency: at day 0, 5, 15 and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: No

STATISTICAL METHODS:

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

5

Sampling time:

28 d

Details on results:

No breakdown products were determined. In a second replicate with the same concentration a biodegradation was only 1 %. Higher test levels could not be obtained because of the limited solubility of the test material in chloroform. This test was conducted from the notifier instead of a test on inherent biodegrability. The German C.A. did not insist on the conduction of the inherent test as the substance also will not be inherent biodegradable for structural reasons.

Results with reference substance:

Sodium benzoate attained 89 % biodegradation within 28 days. Oxygen depletions in the inoculated and non-inoculated control series were within the prescribed limits.

Table 1: Dissolved Oxygen Measurement (mgO2/L)

Treatment		Day
		0	5	15	28
Dilution water, no inoculum	R1	8.50	8.40	8.35	8.20
	R2	8.50	8.40	8.30	8.25
	Mean	8.500	8.400	8.325	8.225
Dilution water, inoculum	R1	8.50	8.35	8.25	8.25
	R2	8.50	8.30	8.25	8.15
	Mean	8.500	8.325	8.250	8.200
Dilution water, inoculum and filter paper	R1	8.50	8.25	8.10	8.05
	R2	8.50	8.30	8.10	8.10
	Mean	8.500	8.275	8.100	8.075
Test Item (2 mg/L) with inoculum	R1	8.50	8.05	8.00	7.90
	R2	8.50	8.10	7.90	7.70
	Mean	8.500	8.075	7.950	7.800
Standard Substance (3 mg/L) with inoculum	R1	8.50	4.80	4.20	3.80
	R2	8.50	4.90	3.90	3.70
	Mean	8.500	4.850	4.050	3.750

Table 2: Mean Oxygen Depletion and Percentage Biodegradation Values

Treatment		Day
		5	15	28
Dilution water, no inoculum	O2 depletion (mgO2/L)	0.100	0.175	0.275
	% Degradation	-	-	-
Dilution water, inoculum	O2 depletion (mgO2/L)	0.175	0.2500	0.300
	% Degradation	-	-	-
Dilution water, inoculum and filter paper	O2 depletion (mgO2/L)	0.225	0.400	0.425
	% Degradation	-	-	-
Test Item (2 mg/L) with inoculum	O2 depletion (mgO2/L)	0.200	0.150	0.275
	% Degradation	4	3	5
Standard Substance (3 mg/L) with inoculum	O2 depletion (mgO2/L)	3.475	4.200	4.450
	% Degradation	70	84	89

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test substance can not be regarded as readily biodegradable.

Executive summary:

In a ready biogradability study according to OECD guideline 301D, an inoculum prepared from non-adapted activated sludge from the aereation stage of the HRC Limited sewage treatment plant (predominantly domestic sewage) was exposed to the test substance. Dissolved oxygen measurements for the test substance and standard substance solutions, together with the inoculated and non- inoculated blank controls, after 0, 5, 15 and 28 days were performed. The positive control sodiumbenzoate attained 89 % degradation within 28 days. The test substance attained 5 % biodegradation after 28 days. Oxygen depletions in the inoculated and non-inoculated control series were within the prescribed limits.

Based on these results, the test substance cannot be considered as readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

sewage inoculum was pre-exposed to test substance

Qualifier:

according to guideline

Guideline:

EU Method C.5 (Degradation: Biochemical Oxygen Demand)

Version / remarks:

as cited in Directive 67/548/EEC

GLP compliance:

yes

Key result

Parameter:

% degradation (CO2 evolution)

Value:

16.5

Sampling time:

28 d

Results with reference substance:

Experimental values (reference substance):
62 % degradation after 10 d
77 % degradation after 14 d
84 % degradation after 24 d
87 % degradation after 28 d

The low solubility of the test material meant the usual SCAS and Zahn-Wellens methods were inappropriate. Instead the modified Sturm test was used with sewage inoculum which had been pre-exposed to the test substance.

Validity criteria fulfilled:

not specified

Interpretation of results:

not inherently biodegradable

Conclusions:

The test substance cannot be regarded as inherently biodegradable.

Executive summary:

Inherent biodegradation has been assessed in accordance with EU method C.5 (ECHA RSS, 2017). The test substance showed 16.5 % degradation until day 28 based on CO2 evolution. No inherent biodegradation can be seen for the test substance.

Description of key information

not readily or inherently biodegradable

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Key Study

In a ready biogradability study according to OECD guideline 301D (Huntingdon, 1989), an inoculum prepared from non-adapted activated sludge from the aeration stage of the HRC Limited sewage treatment plant (predominantly domestic sewage) was exposed to the test substance. Dissolved oxygen measurements for the test substance and standard substance solutions, together with the inoculated and non- inoculated blank controls, after 0, 5, 15 and 28 days were performed. The positive control sodiumbenzoate attained 89 % degradation within 28 days. The test substance attained 5 % biodegradation after 28 days. Oxygen depletions in the inoculated and non-inoculated control series were within the prescribed limits.

Based on these results, the test substance cannot be considered as readily biodegradable.

 

Supporting study

The above result is further supported by the results of an inherent biodegradation study according to EU method C.5 (ECHA RSS, 2017). Although the sewage sludge was pre-exposed to the test substance only 16.5 % biodegradation was observed until study day 28. Thus, the test substance cannot be regarded as biodegradable.