N-(4-{[3-(dimethylamino)propyl]sulfamoyl}phenyl)-2-[(2-methoxy-4-nitrophenyl)diazenyl]-3-oxobutanamide

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:

10 December 2008 to 14 January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-{[3-(dimethylamino)propyl]sulfamoyl}phenyl)-2-[(2-methoxy-4-nitrophenyl)diazenyl]-3-oxobutanamide

EC Number:

700-222-4

Cas Number:

1065519-44-9

Molecular formula:

C22H28N6O7S

IUPAC Name:

N-(4-{[3-(dimethylamino)propyl]sulfamoyl}phenyl)-2-[(2-methoxy-4-nitrophenyl)diazenyl]-3-oxobutanamide

Details on test material:

- Name of test material (as cited in study report): No. 408 yellow
- Physical state: fine powder
- Analytical purity: 98.6%
- Purity test date: 05 November 2008
- Lot/batch No.: 081105-1
- Expiration date of the lot/batch: 04 November
- Storage condition of test material: room temperature
- Other: solubility in water: 0.38 mg/l

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): activated sludge was collected form one of the return lines at Burley Menston sewage treatment works (West Yorkshire, UK).
- Preparation of inoculum for exposure: the suspended solids concentration of the activated sludge was determined by filtering a subsample (25 ml) through a pre-dried and pre-weighed glass microfibre filter (Whatman GF/C). The filter and retained solid were then dried in an oven and re-weighed. The weight of the sludge solids was determined from difference in the weight before and after drying.
- Concentration of sludge: the medium was inoculated with activated sludge at 90 mg suspended solids/L to provide a nominal final solids concentration of 30 mg/L in each test vessel.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

15 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: A test medium concentrate was prepared in ultrapure water contining 30 ml solution (a) and 3 ml of each of solutions (b), (c) and (d). Solutions (a) to (d) were prepared as follows:
(a) potassium dihydrogen phosphate (8.50 g, BDH, AnalaR), dipotassium dihydrate (33.40 g, BDH, AnalaR), ammonium chloride (0.50 g, Sigma, 99.5%), all dissolved in and made upt to 1 l within reverse-osmosis water
(b) calcium chloride dihydrate (36.40 g, BDH, AnalaR), dissolved in and made up to 1 l with reverse-osmosis water
(c) magnesium sulphate heptahydrate (22.50 g, BDH, AnalaR), dissolved in and made up to 1 l with reverse-osmosis water
(d) ferric chloride hexahydrate (0.25 g, Fluka, ACS) and one drop of concentrated hydrochloric acid (BDH, AnalaR), dissolved in and made up to 1 l with reverse-osmosis water
- Test temperature: 22 +/- 2 °C (however, on three occasions the recorded temperature was 19, 16 and 17 °C, for 9, 4 and 4 h respectively. These deviations are not considered to have an impact on the scientific integrity of the study because the degradation of the reference substance was as would be expected and was sufficient to satisfy the appropriate validity criterion)
- pH: 7.38 - 7.62
- Aeration of dilution water: yes
- Suspended solids concentration: 90 mg/l
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: sealed, aerated vessels containing 3 l medium, connected to a series of three traps contining aqueous barium hydroxide (normally 0.0125M)
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: vessels were sealed and aerated by a cylinder of nominally CO2-free air (Air Products).
- Measuring equipment: the air flow was regulated in two stages. Initial control was provided by a gas regulator and the air flow to each vessel controlled by individual needle valves. Measurements were made with a bubble flow meter and stopwatch, at intervals not exceeding seven days, of the flow rate exiting each test vessel. Adjustments were made as necessary to maintain a flow rate of ca. 50 ml per minute.
- Test performed in closed vessels due to significant volatility of test substance: no, vessels were sealed due to air supply
- Details of trap for CO2 and volatile organics if used: at appropriate intervals, the air supply to each vessel was stopped and the trap bottle nearest to the test vessel was removed for sampling. The remaining two bottles of the series were moved towards the test vessel and a fresh trap bottle was placed on the end of the series. Once the series of trap bottles were connected to the test vessel the air supply was restarted. The initial barium hydroxide stock concentrations and the residual concentrations in detached trap bottles were determined by titration against hydrochloric acid (nominally 0.05 M) using 0.5% ethanolic phenolphthalein indicator solution. Titrations were performed on 20 ml trap solution volumes until two matching (+/- 0.1 ml) titres were obtained.
Evolved CO2 from the vessels was trapped in the barium hydroxide traps by formation of a barium carbonate precipitate. This resulted in a decrease in the concentration of barium hydroxide. Consequently, the amount of evolved CO2 was calculated from the decrease in the barium hydroxide concentration, determined by the titration against hydrochloric acid.
On day 28 of the test, each culture vessel was opened and 1 ml concentrated hydrochloric acid added. The vessels were then reconnected to the series of trap bottles and aeration continued until the following day. The acidification and aeration procedure drives off generated carbon dioxide remaining in solution.
Final sampling and titrations were carried out on Day 29, when all of the traps in each series were sampled.


SAMPLING
- Sampling frequency: day 2, 3, 6, 8, 10, 14, 17, 20, 24, 28, 29


CONTROL AND BLANK SYSTEM
- Inoculum blank: duplicate vessels contained inoculated mineral salts medium
- Toxicity control: a single vessel was prepared with inoculated mineral salts medium, test and reference substances (sodium benzoate)
- Other: reference substance: duplicate vessels contained inoculated mineral salts medium and sodium benzoate


STATISTICAL METHODS: theoretical CO2 yields of carbon dioxide (TCO2 in mg) from cultures containing the test and/or reference substances was calculated as follows:
TCO2 = Dabs x Pc x 3.667
where:
Dabs: the absolute dose i.e.: the amount (mg) of test or reference substance added to the culture
Pc: the percentage carbon content of the test or reference substance
3.667: the weight (mg) of CO2 produced from 1 mg of carbon
The theoretical CO2 yield for the quantity of No. 408 yellow applied to the test vessels and toxicity control vessel was 165 mg CO2. Theoretical yields for the quantities of applied sodium benzoate in the toxicity control vessel and reference vessels were also 165 mg CO2. The combined theoretical yield from the toxicity control vessel was 330 mg CO2. The purpose of this control was not to assess the extent of degradation of the entire mixture, but instead to assess the impact of the presence of the test substance on the degradation of the reference material. The theoretical yield in this case was, therefore, limited to the 165 mg CO2 expected from the sodium benzoate alone.
Biodegradation (Dt) of the reference substance and of No. 408 yellow expressed in terms of percentage theoretical CO2 yield was calculated by applying the formulat:
Dt = cumulative mg CO2 produced at time (t) / 165 x 100
All cumulative CO2 values were corrected for the mean CO2 generated by the blanks.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

BOD5 / COD results

Results with reference substance:

The mean percentage biodegradation in the sodium benzoate reference vessels had reached 75% by day 14 and 89% by day 28. The validity criterion of 60% biodegradation at 14 days was therefore met.

Any other information on results incl. tables

Table 1. CO2 Evolution

Test replicate	Evolved CO2(mg)
	Day 2	Day 3	Day 6	Day 8	Day 10	Day 14	Day 17	Day 20	Day 24	Day 28	Day 29*
Blank control (replicate 1)	10.5	15.6	29.2	40.0	48.7	58.3	63.4	67.7	70.1	73.8	78.2
Blank control (replicate 2)	9.9	15.2	29.3	40.7	49.9	59.4	64.6	68.7	72.7	76.3	80.0
Test substance (replicate 1)	9.8	13.6	23.0	29.6	34.0	39.5	42.7	45.8	49.0	52.5	55.8
Test substance (replicate 2)	10.5	14.6	24.1	31.0	35.7	41.0	44.4	47.7	51.0	54.3	58.2
Reference substance (replicate 1)	67.3	90.8	124.7	146.8	163.8	183.1	193.6	201.9	209.7	217.5	224.6
Reference substance (replicate 2)	68.4	92.8	126.4	148.0	165.1	183.7	194.4	202.9	210.8	218.9	226.2
Toxicity control	62.4	84.5	114.6	134.3	147.0	160.5	169.3	176.2	182.2	188.1	194.3

* Day 29 refers to the day that titration of trap contents from acidified vessels was performed. Actual acidification was performed on Day 28

Note: Values for test substance, reference substance and toxicity control vessels are not corrected for blank CO2

Table 2. Biodegradation as a Percentage of Theoretical CO2 Yield

Test replicate	Percentage Biodegradation (%)
	Day 2	Day 3	Day 6	Day 8	Day 10	Day 14	Day 17	Day 20	Day 24	Day 28	Day 29*
Test substance (replicate 1) Test substance (replicate 2)	0   0	0   0	0   0	0   0	0   0	0   0	0   0	0   0	0   0	0   0	0   0
Mean	0	0	0	0	0	0	0	0	0	0	0
Reference substance (replicate 1) Reference substance (replicate 2)	35   35	46   47	58   59	65   65	69   70	75   76	79   79	81   82	84   84	86   87	88   89
Mean	35	46	58	65	70	75	79	81	84	87	89
Toxicity control	32	42	52	57	59	62	64	65	67	69	70

* Day 29 refers to the day that titration of trap contents from acidified vessels was performed. Actual acidification was performed on Day 28

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed