Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

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:

29 March 2017 - 28 April 2017

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)

Version / remarks:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 03 November 2015

Specific details on test material used for the study:

- Solubility in water: not indicated
- Stability in water: stable

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: municipal sewage treatment plant Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage: not applicable, the freshly obtained sludge was used immediately.
- Preparation of inoculum for exposure: the sludge was allowed to settle (31 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium. The day before the start of the test mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
- Concentration of sludge: 3.6 g/L
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

24 mg/L

Based on:

test mat.

Initial conc.:

12 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral medium according to OECD 301
- Test temperature: 22.2 - 23.7°C
- pH: between 7.4 and 7.9 in different test bottles throughout the test
- pH adjusted: no
- Aeration of dilution water: not indicated; tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
- Continuous darkness: yes
- Other: The test solutions were continuously stirred. Furthermore, the test medium was daily swirled around since the test item tended to float on the water surface.

TEST SYSTEM
- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: synthetic air (CO2 < 1 ppm) sparged through scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Measuring method: produced CO2 reacted with barium hydroxide Ba(OH)2 in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl.

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, containing only inoculum (2 bottles)
- Abiotic sterile control: no
- Toxicity control: yes, containing test item, reference item and inoculum (1 bottle).
- Other: positive control: containing reference item and inoculum (1 bottle).

STATISTICAL METHODS: no statistics were used

TEST CONCENTRATIONS:
Test substance (added as weighed amounts to the bottles): bottle A: 24.04 mg/L test substance; bottle B: 23.90 mg/L test substance
Reference control: 40 mg/L reference substance
Toxicity control: 24 mg/L test substance and 40 mg/L reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

Purity: 99.5%

Key result

Parameter:

% degradation (CO2 evolution)

Value:

13

Sampling time:

28 d

Remarks on result:

other: mean of duplicate bottles tested

Details on results:

- The relative biodegradation values were 14% and 11% biodegradation of the test substance (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
- In the toxicity control, more than 25% biodegradation occurred within 14 days (39%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
- Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve
- The theoretical CO2 production of the test substance was calculated to be 1.84 mg CO2/mg and that of the reference substance was calculated to be 1.07 mg CO2/mg.

Results with reference substance:

85% within 14 days

For details, see attached tables in 'illustration' section.

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks' section for details.

Interpretation of results:

not readily biodegradable

Conclusions:

A ready biodegradation test (Modified Sturm test), performed according to OECD 301B and GLP principles, showed that D&C Red 6 was not readily biodegradable.

Executive summary:

In a Modified Sturm Test, according to OECD guideline 301 B and GLP principles, D&C Red 6 was assessed for its ready biodegradability. The test substance was tested in duplicate at a target concentration of 24 mg/L, corresponding to 12 mg TOC/L. The exposure period was 28 days and two inoculum blanks, a positive control and a toxicity control were included. The test substance was not sufficiently soluble to prepare an aqueous solution of 1 g/L. Therefore, weighed amounts were directly added to medium containing sludge and Milli-RO water was added. After vigorous mixing, the suspension was added to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface. CO2 measurements showed that the test substances biodegraded for 14% and 11%, in the duplicate bottles measured. Because the criterion for ready biodegredability was not met, D&C Red 6 was concluded to be not readily biodegradable under the conditions of this test.

The test substance was found not to inhibit microbial activity and all validity criteria were met, thus the study was considered to be valid.

Description of key information

In a Modified Sturm Test, according to OECD guideline 301 B and GLP principles, D&C Red 6 was assessed for its ready biodegradability. The test substance was tested in duplicate at a target concentration of 24 mg/L, corresponding to 12 mg TOC/L. The exposure period was 28 days and two inoculum blanks, a positive control and a toxicity control were included. The test substance was not sufficiently soluble to prepare an aqueous solution of 1 g/L. Therefore, weighed amounts were directly added to medium containing sludge and Milli-RO water was added. After vigorous mixing, the suspension was added to the test medium. The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms. Furthermore, the test medium was daily swirled around, since the test item tended to float on the water surface. CO2 measurements showed that the test substances biodegraded for 14% and 11%, in the duplicate bottles measured. Because the criterion for ready biodegredability was not met, D&C Red 6 was concluded to be not readily biodegradable under the conditions of this test.

The test substance was found not to inhibit microbial activity and all validity criteria were met, thus the study was considered to be valid.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater