4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-21 - 2004-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The test was performed according to relevant guidelines and compliant to GLP. The results are plausible and well documented.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Ordinance relating to Good Laboratory Practice, adopted February 2'1, 2000 [RS 813.016.5]. based on OECD-GLP 1997 (C(97) 186/Final).

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione and 5,12-dihydro-2-methylquino[2,3-b]acridine-7,14-dione and 5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

909-082-0

Cas Number:

938065-79-3

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of 5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione and 5,12-dihydro-2-methylquino[2,3-b]acridine-7,14-dione and 5,12-dihydroquino[2,3-b]acridine-7,14-dione

Specific details on test material used for the study:

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

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During holding, the sludge was aerated at room temperature until use. Prior to use, the sludge was diluted with test water to a concentration of 1 g per liter (dry weight basis). Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

29 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

ca. 20 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

ca. 15.7 mg/L

Based on:

other: TOC (total organic carbon)

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparation of Test Flasks
One day before test start (Day -1), between 2400 and 3000 mL of untreated test medium was filled into 5-liter flasks (amber glass). To each flask (except for the abiotic control and the abiotic control blank), 90 mL of activated sludge inoculum was added.
For the abiotic control and the abiotic control blank, the untreated test medium was poisoned with mercury dichloride at a concentration of 10 mg/L (3 mL of a stock solution containing 10 g HgCl2/L filled up to 3 liters with test medium).
The test media were aerated ovemight with CO2-free air to purge the system of carbon dioxide.
On the following day (Day 0), defined amounts of the test item were directly weighed into the test flasks. No emulsifiers or solvents were used.
The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 770 mg sodium benzoate per 100 mL test water (purged with CO2-free air) was prepared. From this, 10 mL aliquots were added to the corresponding test flasks.
The test flasks were made up to a volume of three liters with test water (purged with CO2-free air). Two absorber flasks, the first one containing 300 mL of 0.05 M NaOH, and the second one containing 200 mL of 0.05 M NaOH, were connected in series to the exit air line of each test flask.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

Preliminary study:

not applicable

Test performance:

No special observations

% Degradation

Parameter:

% degradation (inorg. C analysis)

Value:

3.2

Sampling time:

28 d

Remarks on result:

other: mean of 3.8 and 2.7

Details on results:

The percent biodegradation of the test item was calculated based on a total carbon content (TOC) of 0.78 mg C/mg test item.
The CO2 production of the test item in the test media was in the range of the inoculum controls.
Consequently, the test item was found not biodegradable under the test conditions within 28 days.
Abiotic control:
No degradation of the test item occurred in the abiotic control under the test conditions.

BOD5 / COD results

Results with reference substance:

The percent biodegradation of the reference item was calculated based on a total carbon content (TOC) of 0.58 mg C/mg sodium benzoate.
In the procedure controls, the reference item degraded by an average of 79% by exposure Day 14, thus confirming the suitability of the activated sludge (>60% degradation by Day 14). By the end of the test (Day 28), the reference item was completely degraded.
BIODEGRADATION IN THE TOXICITY CONTROL
The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the total carbon content (TOC) of the test item and the reference item.
Biodegradation in the toxicity control showed a similar course over the 28-day exposure period, when compared to the two procedural controls, containing the reference item only. Within 14 days of exposure, biodegradation amounted to 36%.
Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms because biodegradation in the toxicity control was >25% within 14 days of incubation.

Any other information on results incl. tables

Biodegradation of test and reference item:

Time (days)	% Degradation
	Test item			Na-benzoate			Toxicity control	Abiotic control
	Flask No.			Flask No.			Flask No.
	1	2	Mean	5	6	Mean	4	3
2	0.6	0.0	0.3	39.0	38.9	38.9	17.6	0.0
5	0.3	0.2	0.3	59.1	64.1	61.6	28.6	-0.4
7	0.4	0.8	0.6	63.0	69.1	66.0	29.8	-1.2
9	-1.0	-0.5	-0.8	67.3	73.1	70.2	31.4	-1.4
12	-0.4	-0.2	-0.3	74.7	79.0	76.8	34.3	-2.0
14	-2.0	-1.3	-1.7	75.8	81.8	78.8	36.4	-3.1
19	0.9	0.7	0.8	86.4	88.6	87.5	39.5	-4.6
23	2.5	1.4	2.0	98.4	97.1	97.8	44.3	-6.0
27	1.8	1.7	1.8	99.4	100.3	99.8	45.2	-6.8
28	3.8	2.7	3.2	98.3	100.7	99.5	45.7	-7.0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

In a ready biodegradability test based upon CO2-evolution (reliability category 1, compliant to GLP) no biodegradation could be observed at the end of the test period (28 days). The reference substance (sodium benzoate) was thoroughly degraded at day 28 and to 79% at day 14 proving the suitability of the activated sludge used. On the other hand, lack of biodegradation was not caused by toxicity of the test item as the toxicity control with both, the test item and the reference substance, showed no inhibition compared to the procedural controls with the reference substance only.

Executive summary:

The test item was investigated for its ready biodegradability in a 28-Day CO2 Evolution (Modified Sturm) Test according to EU Commission Directive 92/69/EEC C.4-C (1992) and OECD Guideline for Testing of Chemicals, No. 301 B (1992). The test item was found to be not biodegradable under the test conditions within 28 days. In the abiotic control, containing the test item and poisoned test medium, no degradation was noted at the end of the 28-day exposure period. In the toxicity control, containing both the test item and the reference item sodium benzoate, no inhibitory effect on activated sludge microorganisms was observed. In the procedure controls, the reference item degraded by an average of 79% by exposure day 14, thus confirming suitability of the activated sludge (>60% degradation by Day 14). By the end of the test (Day 28), the reference item was completely degraded.