4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

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:

Experimental start date: 9 November 2001. Experimental completion date: 20 December 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: ST463
Chemical name: 4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-
hydroxynaphthalene-2-carboxamide
Lot number: 6558
Expiry date: October 2002
Purity: 99.9%
Appearance: Magenta powder
Storage conditions: Room temperature
Date received: 15 October 2001

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A sample of activated sludge was collected from Oakley sewage treatment works, which treats predominantly domestic waste. Aliquots (25 ml) of a homogenised sample were filtered through dried (approximately 105°C) and pre-weighed Whatman GFC filter papers. The filters were dried for at least one hour, allowed to cool and re-weighed. The solids level in the sludge was determined and then an appropriate volume used to inoculate control and test vessels to give a final suspended solids concentration of 30 mg/L.

Duration of test (contact time):

29 d

Initial conc.:

10 other: mgC/l

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Air-saturated ultrapure water was added to each of six, five-litre clear glass culture bottles followed by the volumes of each of the stock solutions required to prepare three litres of mineral salts medium. Each culture bottle was then inoculated with activated sludge (30 mg solids/L) and a magnetic stirring bar was added. The clear-glass culture bottles were then wrapped in black polythene in order to protect their contents from light for the duration of the test. The bottles were stoppered and swirled to mix the contents and each was then placed on an electrically-operated magnetic stirrer. The mixtures were aerated overnight with a supply of air that had been treated to remove carbon dioxide by passing it through cylinders containing fused calcium chloride, Carbosorb AS and a trap containing 1M sodium hydroxide solution.

The results of preliminary solubility trials showed that ST463 was insufficiently soluble to allow the preparation of suitable aqueous and organic solvent stock solutions. Therefore, at test initiation on Day 0, appropriate weights of ST463 (44.1 mg) were added to welled glass slides, and added directly to appropriate culture vessels containing inoculated MSM. Glass slides without the test substance were added to control and reference vessels. The reference substance sodium benzoate was added as an aqueous stock solution (1.72 g/L) to one bottle containing the test substance and to one containing inoculated mineral salts medium alone.

Since the test substance was not added to test cultures from a stock solution or suspension it was not necessary to conduct total inorganic and organic carbon measurements. Any inclusion of traces of inorganic carbon was considered to have occurred in the test and the blank-control cultures, so the precision of the test was not considered to have been affected.

The initial pH of each culture was then determined in situ. The final volume of the cultures was assumed to be 3.0 litres. Each vessel was then fitted with a stopper holding an air inlet tube reaching approximately 10 cm below the liquid surface and an air outlet just below the stopper.

The vessels contents were continuously flushed for 29 days with treated air. The air outlet from each vessel was connected to three Dreschel bottles in series, each containing 0.025N, nominal barium hydroxide (100 ml).

The residual concentrations of barium hydroxide in the bottles nearest to the test vessels were determined at intervals by duplicate titration of 20 mL samples with hydrochloric acid (0.05N), using phenolphthalein indicator.

Following the removal of the first Dreschel bottle in a series, the second was connected to the test vessel, and a bottle containing fresh barium hydroxide was connected to the outlet of the bottle at the end of the series.

The biodegradation of the reference substance in the mixture containing the test and reference substance was calculated to confirm that the test substance was not inhibitory to the activity of the microbial inoculum. When the level of biodegradation of sodium benzoate achieved the pass level for ready biodegradability (> 60%), the pH was measured and the treatment was terminated.

On Day 28 of the test, titrations were undertaken and the final pH of each culture was determined in situ. Concentrated hydrochloric acid (1 ml) was then added to each vessel to drive off dissolved inorganic carbon.

The contents of the vessels were aerated overnight and the final titrations carried out on Day 29.

The rate of air flow through the apparatus was determined at intervals during the test.

The minimum and maximum temperature of the test area and of a three-litre volume of water held in a culture bottle under test conditions was recorded daily using a thermometer.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

29 d

Details on results:

Mean cumulative CO2 production by the mixtures containing ST463 was equivalent to 1 % of the TC02 by the end of the test on Day 29.

The pH of all test and control mixtures ranged from 7.5 to 7.6 at the start of the test and ranged from 7.4 to 7.5 at the end .

The rate of air flow during the test ranged from 30 to 75 ml/minute .

The temperature of a three-litre volume of water held under test conditions ranged from 21.8°C to 23.6°C over the test period.

Results with reference substance:

Cumulative C02 production in the controls (74.8 and 71.0 mgC02) was within the acceptable range for this assay system (recommended maximum for a three litre culture = 120 mgC02) . The degradation of sodium benzoate was rapid and had achieved 62 % of its TC02 after 6 days and 87 % after 29 days.

The degradation of sodium benzoate was also rapid in the presence of ST463 and had achieved 65 % of its TCO2 after 6 days. This test vessel was terminated on Day 6 of the study ; the final pH of the culture was 7.7.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The mean cumulative CO2 production by mixtures containing ST463 at 10 mgC/L was negligible and had achieved, at most, 1% of the TCO2 by the end of the test on Day 29.
Substances are considered to be readily biodegradable in this test if CO2 production is equal to or greater than 60% of the theoretical value within ten days of the level achieving 10%. ST463 cannot,
therefore, be considered to be readily degradable.

Executive summary:

The ready biodegradability of ST463 was assessed in the CO₂ Evolution test (Modified Sturm test, Procedure C.4-C of the Annex to Directive 92/69/EEC; OECD Procedure 301B).

ST463 was added to two vessels containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 10 mgCarbon[C]/L. Two control vessels contained inoculated mineral salts medium alone, and one contained inoculated mineral salts medium plus the reference substance sodium benzoate (10 mgC/L). An additional mixture containing sodium benzoate (10 mgC/L) and ST463 (10 mgC/L) was established in order to assess the potential inhibitory effects of the test substance on the microbial inoculum.

Test, control and reference mixtures were aerated for 29 days with air that had been treated to remove carbon dioxide (CO₂). The CO₂ produced by each culture was trapped in a series of Dreschel bottles containing barium hydroxide, which were connected to the outlet from each test vessel. The residual

barium hydroxide was determined at intervals by titration.

The pH of control, reference and test mixtures was measured at the start of the test and after 28 days. The pH of the test plus reference mixture was measured at the start of the test and on the day of its termination (Day 6).

Sodium benzoate had been biodegraded by 62% after 6 days and 87% after 29 days in the absence of ST463, and by 65% after 6 days in its presence which confirmed that ST463 was not inhibitory to the activity of the microbial inoculum. Cumulative levels of CO₂ production in the controls after 29 days

(74.8 and 71.0 mgCO₂) were within the acceptable range for this assay system (recommended maximum = 120 mgCO₂ for a three-litre culture). These results confirm that the inoculum was viable and that the test was valid.

Mean cumulative CO₂ production by mixtures containing ST463 was negligible and had achieved, at most, 1% of the theoretical value (TCO₂, 110.1 mgCO₂) by the end of the test on Day 29.

Substances are considered to be readily biodegradable in this test if CO₂ production is equal to or greater than 60% of the theoretical value within 10 days of the level achieving 10%. ST463 cannot, therefore, be considered to be readily biodegradable

Description of key information

The ready biodegradability of the test substance was assessed in the CO₂Evolution test (Modified Sturm test, Procedure C.4-C of the Annex to Directive 92/69/EEC; OECD Procedure 301B).

The test substance was added to two vessels containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 10 mgCarbon[C]/L. Two control vessels contained inoculated mineral salts medium alone, and one contained inoculated mineral salts medium plus the reference substance sodium benzoate (10 mgC/L). An additional mixture containing sodium benzoate (10 mgC/L) and test substance (10 mgC/L) was established in order to assess the potential inhibitory effects of the test substance on the microbial inoculum.

Test, control and reference mixtures were aerated for 29 days with air that had been treated to remove carbon dioxide (CO₂). The CO₂ produced by each culture was trapped in a series of Dreschel bottles containing barium hydroxide, which were connected to the outlet from each test vessel. The residual

barium hydroxide was determined at intervals by titration.

The pH of control, reference and test mixtures was measured at the start of the test and after 28 days. The pH of the test plus reference mixture was measured at the start of the test and on the day of its termination (Day 6).

Sodium benzoate had been biodegraded by 62% after 6 days and 87% after 29 days in the absence of test substance, and by 65% after 6 days in its presence which confirmed that the test substance was not inhibitory to the activity of the microbial inoculum. Cumulative levels of CO₂production in the controls after 29 days (74.8 and 71.0 mgCO₂) were within the acceptable range for this assay system (recommended maximum = 120 mgCO₂for a three-litre culture). These results confirm that the inoculum was viable and that the test was valid.

Mean cumulative CO₂production by mixtures containing test substance was negligible and had achieved, at most, 1% of the theoretical value (TCO₂, 110.1 mgCO₂) by the end of the test on Day 29.

Substances are considered to be readily biodegradable in this test if CO₂ production is equal to or greater than 60% of the theoretical value within 10 days of the level achieving 10%. The test substance cannot, therefore, be considered to be readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater