Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]

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:

16 October 2018 to 12 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Incorrect molecular wieght provided, values adjusted, no impact on study

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Incorrect molecular wieght provided, values adjusted, no impact on study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]

EC Number:

274-354-1

EC Name:

Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]

Cas Number:

70161-18-1

Molecular formula:

C47H36N6O12S4.2Na

IUPAC Name:

disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]

Test material form:

solid: crystalline

Specific details on test material used for the study:

Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 15 October 2018 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1-Hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.1 g/L prior to use.

Duration of test (contact time):

> 28 - < 56 d

Initial test substance concentration

Initial conc.:

9.9 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preparation of Test System
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper*.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a filter paper* to give a final concentration of 10 mg carbon/L.
c) The test item on a filter paper*, in duplicate, in inoculated mineral medium to give a final concentration of 9.9 mg carbon/L.
d) The test item on a filter paper* plus the reference item in inoculated mineral medium to give a final concentration of 19.9 mg carbon/L to act as a toxicity control (one vessel only).
A filter paper with dimethylformamide evaporated to dryness was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 22 °C and 24 °C for 56 days, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 42.9 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter and adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all of the vessels being adjusted to 3 liters by the addition of mineral medium, which had been purged overnight with CO2-free air. The inoculum control vessels were prepared in a similar manner without the addition of test item or reference item.

The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L.

Results and discussion

Preliminary study:

Preliminary Solubility Work
Information provided by the Sponsor indicated that the test item was soluble in water. However preliminary work indicated that the test item was not soluble in mineral medium. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation

Test performance:

The IC values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. TC and IC values in the culture vessels on Day 0 are given in Table 3. The pH values of the test preparations on Days 0 and 56 are given in Table 4. Observations made on the contents of the test vessels are given in Table 5.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

56 d

Details on results:

The total CO2 evolution in the inoculum control vessels on Day 28 was 27.43 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.
Acidification of the test vessels on Day 56 followed by the final analyses on Day 57 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 57 samples originated from dissolved CO2 that was present in the test vessels on Day 56 and hence the biodegradation value calculated from the Day 57 analyses is taken as being the final biodegradation value for the test item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 57 showed an increase in all replicate vessels with the exception of inoculum control Replicate 1 and procedure control Replicate 2.
The IC analysis of the samples from the second absorber vessels on Day 57 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The test item attained 0% biodegradation after 28 and 56 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Statistical analysis of the Day 57 IC values for the inoculum control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the inoculum control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.
The toxicity control attained 30% biodegradation after 14 days and 29% biodegradation after 28 and 56 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. The slight decrease in biodegradation between Days 14 and 28 and 56 was considered to be due to sampling and/or analytical variation.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 68% biodegradation after 14 days with greater than 60% degradation being attained in a 10-day window. After 28 days 76% biodegradation was attained with 81% biodegradation after 56 days. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item attained 0% biodegradation after 28 and 56 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B

Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)), in accordance with GLP.

The test item attained 0% biodegradation after 28 and 56 days and therefore cannot be considered to be readily biodegradable under the conditions of OECD Guideline No. 301B