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Biodegradation in water: screening tests

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Reference
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
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
2008 (2008/440/EC)
Deviations:
yes
Remarks:
An optional toxicity control as suggested by OECD guideline 301 F was not performed. The validity of the study is not adversely affected.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
An optional toxicity control as suggested by OECD guideline 301 F was not performed. The validity of the study is not adversely affected.
Principles of method if other than guideline:
Deviations from guidelines, study plan and standard operating procedures :

An optional toxicity control as suggested by OECD guideline 301 F was not performed. The validity of the study is not adversely affected.

An apparent decrease in biodegradation on one of the flasks containing reference substance (flask 4) can be attributed to a minor leakage. As the validity criteria are fulfilled on day 28, at the end of the standard test, the validity of the study is not adversely affected.
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (Bois-de-Bay, Satigny, Switzerland) was used.

The sludge was collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.

Determination of the dry weight of suspended solids :
The dry weight of suspended solids is determined by taking two 50 ml samples of the homogenised sludge, evaporating water on a steam bath, drying in an oven at 105 - 110°C for two hours and weighing the residue.

Activated Sludge :
Dry weight of suspended solids: 4.67 g/l, diluted to 2.62 g/L

To obtain a concentration of 30 mg/L (dry weight) in 437 ml total volume, 5.00 mL of sludge (inoculum) was added to 432 mL of mineral medium.
Duration of test (contact time):
61 d
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The method used is basically the one described under No. 301 F in the OECD Guidelines for Testing of Chemicals.

A measured volume of inoculated mineral medium, containing a known concentration of test substance as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (± 1°C) for up to 28 days. Evolved carbon dioxide is absorbed in sodium hydroxide pellets. The consumption of oxygen is determined by measuring the pressure drop in the respirometer flask. The Biological Oxygen Demand (BOD), amount of oxygen taken up by the microbial population during biodegradation of the test chemical (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidized to carbon dioxide, hydrogen to water and nitrogen to ammonium, nitrite or nitrate).

Water :
The water used during this study is ultrapure water, containing less than 5 ppb total organic carbon, produced by using a Millipore Direct-Q 3 UV purification system.

Stock solutions of mineral components :
The following stock solutions were prepared -

Solution A:

KH2PO4 8.5 g
K2HPO4 21.75 g
Na2HPO4 · 2 H2O 33.4 g
NH4Cl 0.5 g

dissolved in water and made up to 1 litre.

Solution B:

CaCl2 27.5 g

dissolved in water and made up to 1 litre.


Solution C:

MgSO4 · 7 H2O 22.5 g

dissolved in water and made up to 1 litre.


Solution D:

FeCl3 · 6 H2O 0.25 g
HCl Conc. one drop

dissolved in water and made up to 1 litre.

Mineral medium :
Prepared by mixing 50 ml of solution A and 2000 ml deionised water, adding 5 ml of each of the solutions B, C and D and making up to 5 litres with deionised water. The pH is measured and if necessary adjusted to 7.4 ± 0.2 with phosphoric acid or potassium hydroxide.

Test substance samples (8.74 mg, corresponding to 20.0 mg/l in 437 ml of test medium) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop. For reference substance samples 43.70 mg (corresponding to 100.0 mg/l in 437 ml of test medium) are weighed in small aluminium boats and added directly to the test flasks of the Oxitop.

Flasks are filled with 432 mL of mineral medium. Samples of test or reference substance are added. Then 5.00 mL of suspended sludge diluted to a concentration of 2.62 g/L dry matter is added. Except when the test substance has an acid or alkaline character, the pH of each flask is not measured but assumed to be the same as the mineral medium, in order not to remove any floating undissolved test substance from the test medium by dipping a glass electrode in it. Neutral test substances, even sodium benzoate, were shown not to affect the pH of the medium by more than 0.1 pH unit. Two sodium hydroxide pellets are placed in the quivers on top of the bottle, and the flasks are closed tightly with the measuring heads. The flasks are allowed to equilibrate to the test temperature. The measurement is started by programming the measuring unit of the Oxitop test flasks, and the test flasks are placed in the temperature controlled cupboard of the Oxitop system. After temperature equilibration, the controller of the instrument starts the data acquisition (time zero of the experiment).

Performance of the test :
Everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked.

At the end of the test period, the pH of each flask is measured again.


Test and reference substances :
Nominal concentrations :
- test substance : 20 mg/L
- reference substance : 100 mg/L

Test temperature and duration :
Test temperature : 20.6°C – 21.4°C
Test duration : 61 days

Oxygen uptakes, as read on the Oxitop controller, are corrected to account for the small differences between actual and nominal concentrations of test and reference substances.
Reference substance:
benzoic acid, sodium salt
Test performance:
Everyday the oxygen consumption of each flask is recorded and correct temperature and stirring are checked.
At the end of the test period (normally 28 days), the pH of each flask is measured again.

Parameter:
% degradation (O2 consumption)
Value:
74
Sampling time:
61 d
Remarks on result:
other: Day 61 BOD average = 74% (74% and 74% in the two flasks.
Key result
Parameter:
% degradation (O2 consumption)
Value:
62
Sampling time:
28 d
Remarks on result:
other: Day 28 BOD average = 62% (66% and 58% in the two flasks).
Details on results:
GR-86-6599 did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

GR-86-6599 undergoes 62% biodegradation after 28 days in the test conditions. The 10-day window criterion is not fulfilled (11% biodegradation on day 4 and 41% on day 14). The 10-day time window criterion was developed on the assumption that a test substance is degraded according to a “standard” growth curve in ready biodegradability tests. The OECD Guidelines for Testing of Chemicals, Section 3. Part 1: Principles and strategies related to the testing of degradation of organic chemicals (adopted 23 March 2006), states that if a substance consists of “constituents with different chain-lengths, degree and/or site of branching or stereo-isomers, even in their most purified commercial forms” and “it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test.” The 10-day time-window should therefore not be applied as a pass / fail criterion for GR-86-6599, which is a mixture of isomers. Thus, GR-86-6599 should be regarded as readily biodegradable according to this test based on the biodegradation percentage of 62% at day 28.

Degradation of sodium benzoate exceeded 40% after 7 days and 65% after 14 days: the activity of the inoculum was thus verified (validity criterion).

The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled. Therefore, the test is considered valid.

Results with reference substance:
Degradation of sodium benzoate exceeded 40% (observed = 72%) after 7 days and 65% after 14 days (observed = 77%): the activity of the inoculum was thus verified (validity criterion).
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
GR-86-6599 did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

GR-86-6599 undergoes 62% biodegradation after 28 days in the test conditions. The 10-day window criterion is not fulfilled (11% biodegradation on day 4 and 41% on day 14). The 10-day time window criterion was developed on the assumption that a test substance is degraded according to a “standard” growth curve in ready biodegradability tests. The OECD Guidelines for Testing of Chemicals, Section 3. Part 1: Principles and strategies related to the testing of degradation of organic chemicals (adopted 23 March 2006), states that if a substance consists of “constituents with different chain-lengths, degree and/or site of branching or stereo-isomers, even in their most purified commercial forms” and “it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test.” The 10-day time-window should therefore not be applied as a pass / fail criterion for GR-86-6599, which is a mixture of isomers. Thus, GR-86-6599 should be regarded as readily biodegradable according to this test based on the biodegradation percentage of 62% at day 28.
Executive summary:

The Ready Biodegradability of GR-86-6599 was determined by the Manometric Respirometry Test.

GR-86-6599 did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.

GR-86-6599 undergoes 62% biodegradation after 28 days in the test conditions.The 10-day window criterion is not fulfilled (11% biodegradation on day 4 and 41% on day 14).The 10-day time window criterion was developed on the assumption that a test substance is degraded according to a “standard” growth curve in ready biodegradability tests. The OECD Guidelines for Testing of Chemicals, Section 3. Part 1: Principles and strategies related to the testing of degradation of organic chemicals (adopted 23 March 2006), states that if a substance consists of “constituents with different chain-lengths, degree and/or site of branching or stereo-isomers, even in their most purified commercial forms” and “it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test.” The 10-day time-window should therefore not be applied as a pass / fail criterion forGR-86-6599, which is a mixture of isomers.

Description of key information

Key study, OECD 301F, GLP, 2017, Klimisch 1:


The Ready Biodegradability of GR-86-6599 was determined by the Manometric Respirometry Test.


 


GR-86-6599 did not inhibit the intrinsic respiration of the inoculum at the test concentration and was therefore considered to be non-toxic to the inoculum at the test concentration.


 


GR-86-6599 undergoes 62% biodegradation after 28 days in the test conditions.The 10-day window criterion is not fulfilled (11% biodegradation on day 4 and 41% on day 14).The 10-day time window criterion was developed on the assumption that a test substance is degraded according to a “standard” growth curve in ready biodegradability tests. The OECD Guidelines for Testing of Chemicals, Section 3. Part 1: Principles and strategies related to the testing of degradation of organic chemicals (adopted 23 March 2006), states that if a substance consists of “constituents with different chain-lengths, degree and/or site of branching or stereo-isomers, even in their most purified commercial forms” and “it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test.” The 10-day time-window should therefore not be applied as a pass / fail criterion forGR-86-6599, which is a mixture of isomers.


 


As such, the test item is considered Readily Bidoegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information