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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
(adopted July 1992)
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Test organisms:
- Type : mixed population of aquatic microorganisms (activated sludge)
- Origin : aeration tank of a wastewater plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)
- Date of collection : 2017-04-8
- Concentration of inoculum : 30 mg/L suspended solids

Pre-treatment of the inoculum:
− Before use, the inoculum was stored for one day at room temperature under continuous shaking with aeration.
− The sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant.
− An aliquot of the wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension (activated sludge) of 3 g dw/L.
− The calculated amount of sludge, needed to achieve 300 mL of this stock suspension, was dissolved in mineral medium and then filled up to a defined end volume.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
I. Pre-treatment of test item:
- 25 mg of test item were weighed on aluminum foil and added to the test flasks, filled with 200 mL of mineral medium
- Afterwards the volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item/L

II. Pre-treatment of the reference compound:
- 25 mg of the reference compound were weighed on aluminum foil and added to the test flasks, filled with 200 mL of mineral medium
- Afterwards the volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg reference compound/L

III. Pre-treatment of the toxicity control:
- 25 mg of test item and 25 mg of the reference compound were weighed on aluminum foil and added to the test flasks, filled with 200 mL of mineral medium
- Afterwards the flask volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item and reference compound/L.

IV. Exposure conditions:
- Test volume : 250 mL
- Test apparatus : OxiTopControl System (WTW)
- Mixing : 1 magnetic stirrer per test vessel
- Incubation time : 28 days
- Incubation temperature : 22 ± 1 °C
Reference substance:
benzoic acid, sodium salt
Remarks:
(Acros Organics, batch no.: A0357641, purity: 99.9 %)
Test performance:
TEST PROCEDURE:
- A suspension of 100 mg/L Solvent Yellow 179 in a mineral medium was stirred in a closed flask and inoculated at a constant temperature (22 ± 1 °C) for up to 28 days under aerobic conditions in the dark
- The consumption of oxygen (BOD) was determined by measuring the drop in pressure in the automated respirometer flasks
- Evolved carbon dioxide was absorbed in sodium hydroxide
- The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of theoretical oxygen demand (ThOD)
- The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without Solvent Yellow 179.
- A reference compound (sodium benzoate) was run in parallel
- A toxicity control (Solvent Yellow 179 and reference compound mixed, one replicate) was run in parallel
- Degradation was followed by the determination of oxygen uptake and measurements were taken at frequent intervals to allow the identification of the beginning and end of biodegradation and the slope of the biodegradation curve; determinations of Solvent Yellow and inoculum blank were carried out in triplicate and of reference compound in duplicate.
- The oxygen uptake was calculated from the readings taken at regular and frequent intervals
- At the end of incubation, the pH was measured
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
7 d
Details on results:
No nitrification was observed in the flasks with test item. The oxygen consumed by nitrification was not determined at the end of the study, because no degradation of the test item was observed.
Key result
Parameter:
ThOD
Value:
2.334 other: mg O2/mg
Results with reference substance:
Degradation of reference compound:
- 85 after 7 days
- 94 after 14 days
- 96 after 21 days
- 97 after 28 days

After 28 days the pH values of different flasks are in the range of 7.5 -8.1.

Validity criteria fulfilled:
yes
Remarks:
(- Ready biodegradation of reference compound ≥ 60 % within 14 days. - In the toxicity control degradation rates > 25 % within 14 days. - Replicates difference < 20 %. - Oxygen uptake of blank inoculum was ≤ 60 mg/L.)
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Within 28 days, a degradation rate of 0 % was determined. Solvent green 28 is considered to be "Not Readily Biodegradable". The reference compound sodium benzoate showed 94 % degradation after 14 days.
Executive summary:

The study was performed to assess the ready biodegradability of solvent green 28. The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.4-D “Manometric Respirometry Test“(2008). This test method is in all essential parts identical with OECD Guideline 301 F (adopted July 1992). A suspension of solvent green 28 in a mineral medium was inoculated and incubated for 28 d under aerobic conditions. During this period, degradation was followed by continuous automated BOD determinations. Within 28 days, a degradation rate of 0 % was determined. Solvent green 28 is considered to be "Not Readily Biodegradable". The reference compound sodium benzoate showed 94 % degradation after 14 days. The used concentration of the solvent green 28 was not toxic to bacteria.

Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Origin: Aeration tank of a municipal biological waste water treatment plant, not adapted, ARA Werdholzli, CH-8048 Zurich
- Pre-Treatment: not pre-conditioned
- Concentration of inoculum: 0.2 g/L dry matter in the final mixture
- Date of collection: 15 May 2003; 9.00 a.m.
Duration of test (contact time):
28 d
Initial conc.:
0.2 g/L
Based on:
DOC
Initial conc.:
50 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST SYSTEM:
- Test unit: 1200 mL closed glass bottle containing a total volume of test solution of 600 ml; aerated with C02-free air and fitted to gas-absorption bottles
containing 120 mL of 0.5 M NaOH
- Test medium: Aerobic mineral salts medium prepared with double distilled water (conductivity: <1.5 µS/cm; DOC: <0.3 mg/L)
- Feed: None, test substance or procedure control as sole organic carbon sources
- Incubation: Temperature-controlled dark room (22 ± 0.5 °C)

Reference substance:
diethylene glycol
Remarks:
49.6 mg/L (as DOC)
Test performance:
TEST PROCEDURE:
- The activated sludge was used immediately after sampling from the treatment plant without adaptation
- Prior to the test the sludge was washed twice with tap water
- The test material was diluted with mineral salts medium to give a final DOC concentration of about 50 mg/L
- For each test series the following number of test flasks was set up: Test suspension (T) 2 replicates; Inoculum blank (B), 2 replicas; Procedure control (R), 1 replicate
- The test vessels were stirred (100 r.p.m.) and aerated with synthetic C02-free air
- The air leaving the individual vessels was passed through gas-absorption bottles filled with NaOH
- lt was assured that during the test the oxygen concentration was > 6 mg/L
- The pHvalue was checked periodically and adjusted to pH 6.5-8.0 with NaOH or H2S04, if necessary
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
100
Sampling time:
4 d
Remarks on result:
other:
Remarks:
Due to the limited water solubility of the test item elimination occurred immediately
Details on results:
Based on the data of the individual DOC determinations the test item reached an elimination of 100 % after 28 days, calculated with respect to the applied theoretical TOC concentration. Due to the limited water solubility the elimination occurred immediately. 100 % elimination was observed after 4 days of incubation.
No biodegradation based on C02 evolution and calculated as % ThC02 was observed.
Results with reference substance:
The positive control, diethyleneglycol, showed 100 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions. The extent of mineralization of the procedure control with diethyleneglycol was 90% based on ThC02.
Validity criteria fulfilled:
yes
Remarks:
The test was considered valid, since the degradation of the reference compound reached more than 70 %within 14 days of incubation.
Interpretation of results:
not inherently biodegradable
Conclusions:
Based on the mineralization data Solvent green 28 is not inherently biodegradable under the conditions of the OECD guideline no. 302, since less than 20 % degradation (mineralization) was attained after 28 days of contact time. However, based on DOC measurements the substance was completely eliminated.
Executive summary:

The objective of this study was to determine the biodegradability under aerobic static conditions using the Zahn-Wellens I/EMPA Test. Activated sludge, mineral nutrients and the test material as the sole carbon source were incubated together in a glass vessel placed on an orbital shaker under controlled conditions. The degradation of the test material was monitored by the determination of the dissolved organic carbon (DOC) at regular time intervals. The ratio of eliminated DOC after each interval to the value 3 hours after the start of the test was expressed as percentage biodegradation. The percentage biodegradation was plotted against time to give the biodegradation curve. The produced C02 was determined at least 3 times during the test as an additional measure of the biodegradation expressed as the percentage of the theoretical value (% ThC02). The biodegradability of Solvent Green 28 exposed to activated sludge of a municipal sewage treatment plant was investigated under aerobic static conditions. At the applied test concentration the test material was only partially dissolved. Based on the data of the individual DOC determinations Solvent Green 28 reached an elimination of 100 % after 28 days, calculated with respect to the applied theoretical TOC concentration. Due to the limited water solubility of Solvent Green 28 DOC elimination occurred immediately. 100 % elimination was observed after 4 days of incubation. The positive control, diethyleneglycol, showed 100 % biodegradation after 14 days of incubation thus confirming suitability of inoculum and test conditions. The test was considered valid, since the degradation of the reference compound reached more than 70 %within 14 days of incubation. No biodegradation of Solvent Green 28 based on C02 evolution and calculated as % ThC02 was observed. The extent of mineralization of the procedure control with diethyleneglycol was 90% based on ThC02. Based on the mineralization data Solvent Green 28 is not inherently biodegradable under the conditions of the OECD guideline no. 302, since less than 20 % degradation (mineralization) was attained after 28 days of contact time. However, based on DOC measurements Solvent Green 28 was completely eliminated.

Description of key information

Within 28 days, a degradation rate of 0 % was determined. Solvent green 28 is considered to be "Not Readily Biodegradable". The reference compound sodium benzoate showed 94 % degradation after 14 days.

In another study on based on the mineralization data Solvent green 28 is not inherently biodegradable under the conditions of the OECD guideline no. 302, since less than 20 % degradation (mineralization) was attained after 28 days of contact time. However, based on DOC measurements Solvent Green 28 was completely eliminated.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The toxicity control exhibited degradation rates > 25 % within 14 days.