Chromate(5-), bis[4-(hydroxy-κO)-7-[2-(2-hydroxy-1-naphthalenyl)diazenyl]-3-[2-[2-(hydroxy-κO)- 3-nitro-5-sulfophenyl]diazenyl]-2-naphthalenesulfonato(4-)]-, sodium (1:5)

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

-Source of inoculum/activated sludge: 'Waterschap de Dommel', 's-Hertogenbosch, the Netherlands.
-Pretreatment: the sludge was kept under continuous aeration untn further treatment. The concentration of suspended solids was 3.5 g/l in the concentrated sludge. Before use, the sludge was allowed to settle for at least 30 minutes and the liquid decantad for use as Inoculum at the amount of 10 ml/I of mineral medium.

Duration of test (contact time):

ca. 28 d

Initial conc.:

15 other: mg TOC/l

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

PREPARATION OF TEST SOLUTIONS
The test substance was quantitatively added to the test media and continuously stirred during the test.

TEST CONDITIONS
- 1 l mineral medium contains: 10 ml of solution (a), 1 ml of solutions (b) to (d) and Milli-Q water.
-Composition of medium:
A) 8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HP04.12H20
0.50 g NH4CI
dissolved in 1 l Milli-Q water, pH 7.4 ± 0.2
B) 22.50 g MgS04.7H20 dissolved in 1 l Milli-Q water.
C) 36.40 g CaCl2.2H20 dissolved in 1 l Milli-Q water.
D) 0.15 g FeCl3 dissolved in 1 l Milli-Q water.
- Milli Q water: Tap-water purified by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges
- Test vessel: 2 litre all-glass brown coloured bottles.
- Barium hydroxide, 0.0125 M: 4 g Ba(0H)2.8H20 per litre Milli-Q water was filtered through filter paper and stored in a sealed vessel to prevent absorption of CO2 from the air
-C02-free air: A mixture of oxygen (21%) and nitrogen (79%) was led through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(0H)2 solution to trap CO2 which might be present in small amounts. The C02-free air was sparged through the scrubbing solutions at a constant rate.
-Test concentration: the test substance was tested in duplicate at 73.6 (A) and 73.8 (B) mg, corresponding to 15 mg TOC/l. The organic carbon content was based on analysis of TC.
-Test temperature: 18.5-24 °C (recorded daily during the test period)
-pH: 7.5 - 7.9 (measured just before the start of the test and at 28 d)
-pH adjusted: no

Preparation of bottles:
-Pre-incubation medium: mineral components, Milli-Q water (ca. 80 % total volume) and inoculum (1 % final volume) were added to each bottle. This mixture was aerated with C02-free air overnight to purge the system of CO2.
-Type and number of bottles: test suspension containing test substance and inoculum (2 bottles), Inoculum blank containing only inoculum (2 bottles), Procedure control containing reference substance (40 mg/l sodium acetate, TOC= 12 mg/l) and inoculum (1 bottle).
-Additional bottles: since no information was available about the inhibition of microbial activity a toxicity control was added containing test substance, reference substance and inoculum (1 bottle).
-Preparation: the test substance and positive control were added to the bottles. The volumes of suspensions were made up in all bottles by the addition of Milli-Q water previously aerated with C02-free air, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(0H)2) were connected in series to the exit air line of each test bottle.
-Start of the incubation: The test was started by bubbling C02-free air through the solution at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).

TEST SYSTEM
-Number of culture flasks/concentration: 2

CONTROL AND BLANK SYSTEM
-Inoculum blank: containing only inoculum (2 bottles)

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

7.7

Sampling time:

28 d

Remarks on result:

other: 73.6 mg test substance

Key result

Parameter:

% degradation (CO2 evolution)

Value:

7.4

Sampling time:

28 d

Remarks on result:

other: 73.8 mg test substance

Details on results:

The Total Carbon content (TC) of the test substance was determined to be 39.6 %. The concentration was 73.6 (A) and 73.8 (B) mg test substance in 2 litres test medium. Hence, the theoretical CO2 production following complete degradation was 106.9 mg per 2 litres for A and 107.2 mg per 2 litres for B.
The total CO2 release in the blank reached a total value of 2.37 mg CO2 per 2 litres of medium.
The difference of duplicate values for degradation of test item was always less than 20%.
In the toxicity control more than 25 % degradation occurred in 14 days (based on ThCO2). Therefore, the test substance was assumed to be not inhibitory.

Results with reference substance:

The positive control contained 80.5 mg sodium acetate (ThC02= 1.073 mg CO2/mg) resulting in a theoretical CO2 production following complete degradation of 86.4 mg per 2 litres. The control substance was degraded 76 % in 14 days and 87 % degradation was reached at the end of the test period.

A deviation the temperature range mentioned in the protocol was noted, but was considered not to have affected the integrity of this study.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this test substance was not readily biodegradable.

Executive summary:

The study was performed to determine the potential for ready biodegradation of the test item in water by the carbon dioxide (CO2) evolution method following the method C.4. (Modified Sturm Test)of EEC-Directive 92/69. The CO2released upon biodegradation of the test item and the reference substance was measured over 28 days. All validation criteria were fulfilled. The control substance was degraded > 60 % in 14 days. The test substance was found to be not inhibitory. The relative degradation values calculated from the measurements performed during the test period revealed no significant degradation of the test substance (7 -8 %).

Therefore the substance was considered to be not readily biodegradable.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

-Source of inoculum/activated sludge: microorganisms from a domestic waste-water treatment plant (ARA Füllinsdorf/Switzerland)
-Preparation of inoculum for exposure: the sludge was centrifuged, the supernatant liquid phase decanted, the solid material resuspended in tap water again centrifuged. This procedure was repeated a further two times. An aliquot of the final sludge suspension was weighed, thereafter dried and the ratio (R) of wet sludge to dry sludge calculated. The amount of wet sludge corresponding to 4 g ± 10 % (dry weight basis) was added to one litre of Soerensen buffer (pH 7). It was kept aerated until use at 20 - 25 °C.
-Concentration of sludge: An amount of sludge from a domestic waste-water treatment plant corresponding to 0.2 g dry material was added per litre final test medium.

Duration of test (contact time):

28 d

Initial conc.:

207 mg/L

Based on:

test mat.

Remarks:

Test No. 1

Initial conc.:

208.5 mg/L

Based on:

test mat.

Remarks:

Test No. 2

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

TEST SYSTEM
-Apparatus: aerated 2.5-1 cylindrical reactor flasks
-Composition of medium:
The stock solutions according to guidelines were:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HP04X 2H2O, 0.5 g NH4CI
dissolved up to 1000 ml bidistilled water. The pH should be 7.4.
b) 22,5 g MgS04X 7H2O dissolved up to 1000 mi bidistilled water.
c) 36.4 g CaCIgX 2H2O dissolved up to 1000 mi bidistilled water.
d) 0.25 g FeCI3 X 6H2O dissolved up to 1000 mi bidistilled water.
In order to avoid having to prepare this solution immediately before use, 1 (one) drop of concentrated HCI or 0.4 g ethylenediaminetetraacetic acid (EDTA, disodium salt) per litre was added.
10 ml of stock solution (a) and one (1) mi of each stock solution (b-d) were added up to 1000 ml bidistilled water.
-Test Concentration: based on pre-test results (raw data were available but the pre-test was not be performed in compliance with the GLP guidelines). The DOC of a solution containing 64 mg test item/ 200 ml bidistilled water was 122.5 mg C/l. Therefore, 414 and 417 mg test item were each filled to a final volume of 2000 ml. The corresponding starting DOC concentrations were 79.8 and 80.0 mg C/l (corrected for the blank). The concentration of the standard compound (Aniline) was 202 mg/ 2 litre (101 mg/l) corresponding to a theoretical amount of 78.2 mg C/l. In addition, an untreated inoculation control was run.

TEST CONDITIONS
-Test temperature: 21.5 - 22.0 °C
-pH: from 7.2 to 7.8
-pH adjusted: no
-Aeration: The flasks were aerated with a flow rate of about 0.5 - 0.7 I/minute, resulting in an oxygen concentration of 8.4 - 9.1 mg O2 per litre.
-Suspended solids concentration:
-Continuous darkness: yes

CONTROL AND BLANK SYSTEM
-Inoculum blank: untreated inoculated sample
-Procedure control: sample with as standard compound.

SAMPLING
At the respective sampling day, water evaporation losses were compensated by adding bidistilledwater. If necessary, deposits on the test vessel were scraped off. Per sampling interval, one sample of about 30 ml was taken per flask and anaiysed for DOC in triplicate.
-Sampling frequency: at day 0 (0 and 3 hours after treatment), 5, 7, 14, 21, 27 and 28 of the incubation period. Before samples for analyses were taken the water evaporation losses were compensated by adding bidistilled water.

Reference substance:

aniline

Remarks:

purity >99.5 %

Key result

Parameter:

% degradation (DOC removal)

Value:

0

Sampling time:

28 d

Results with reference substance:

The reference compound, aniline, was readily biodegraded within 7 days by 94 %. At the end of the test, aniline was degraded by 100 %.

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

not inherently biodegradable under the test conditions

Executive summary:

The substance was investigated for its biodegradability in the 28 days Zahn Wellens/EMPATest, according to the OECD guideline 302B. The DOC concentration of test item remained unchanged throughout the entire test. The standard, aniline, was degraded within 7 days of exposure by 94 %. At the end of the test, aniline was degraded by 100 %.

Thus, substance was found to be not degradable under the present test conditions.

Description of key information

Not ready biodegradable

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Biodegradation in water of the substance was evaluated by screen tests. The studies were performed to determine the potential for ready and inherent biodegradability, according to EEC directive 92/69, Part C.4. (Modified Sturm Test) and to the OECD guideline 302B respectively.

The test substance is considered as not readily biodegradable in water.