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Environmental fate & pathways

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:
The study was conducted between 3 August 2016 and 21 November 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
An optional toxicity control as suggested by OECD guideline 301F was not performed. The validity of the study is not adversely affected.
Deviations:
no
Remarks:
An optional toxicity control as suggested by OECD guideline 301F was not performed. The validity of the study is not adversely affected.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Lot No.: SC00012329
Aspect: Yellow to brown yellow liquid

Due to the complex composition of the test substance, the Biological Oxygen Demand (BOD) was expressed as a percentage of Chemical Oxygen Demand (COD). The COD of the batch tested was determined to be 2.550 mgO2/mg according to the DIN 38414 method.
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly 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 is 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.
Dry weight of suspended solids: 9.52 g/I, diluted to 2.62 g/l
To obtain a concentration of 30 mg/I (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):
67 d
Initial conc.:
30 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
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
Na2H PO4.2H2O: 33.4 g
NH4CI: 0.5 g
dissolved in water and made up to 1 litre.

Solution B:
CaCl2: 27.5g
dissolved in water and made up to 1 litre.

Solution C:
MgSO4•7H2O: 22.5g
dissolved in water and made up to 1 litre.

Solution D:
FeCI3.6H2O: 0.25g
HCI 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.

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.

Preparation of the flasks
Test substance samples (13.10 mg, corresponding to 30 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 43.7mg (corresponding to 100mg/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, 5ml 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 flaks, and the test flask are plaved in the termperature 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
Every day 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.

Test temperature: 20.8 - 21.7°C
Test duration: 67 days
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
41
Sampling time:
28 d
Remarks on result:
other: mean value
Parameter:
% degradation (O2 consumption)
Value:
56
Sampling time:
67 d
Remarks on result:
other: mean value
Key result
Parameter:
% degradation (O2 consumption)
Value:
62
Sampling time:
67 d
Remarks on result:
other: individual replicate
Details on results:
- 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.
- Degradation of sodium benzoate exceeded 40 % after 7 days and 65 % after 14 days: the activity of the inoculumwas thus verified (validity criterion).
- The repeatability validity criterion (not more than 20% difference between replicates) was fulfilled. Therefore, the test is considered valid.
- Citrathal Concentrate S TW undergoes 41% biodegradation after 28 days (56% after 67 days) in the test conditions. At the test concentration Citrathal Concentrate S TW did inhibit the intrinsic respiration of the inoculum by more than 20% on day 1 of the test. Toxicity towards the inoculum at test concentration can therefore not be excluded.
Citrathal Concentrate S TW should be regarded as not readily biodegradable according to this test.
- Individual values for the two replicates at 67 days were 50 and 62%. The latter meets the pass criterion for inherently and ultimately biodegradable.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
Citrathal Concentrate S TW undergoes 41% biodegradation after 28 days, which is insufficient to consider the substance as readily biodegradable. Since a plateau was not reached, the test was extended. The biodegradation continued reaching a mean level of 56% after 67 days in the test conditions. The individual values for the two replicates were 50 and 62%. The latter meets the pass criterion for inherently and ultimately biodegradable.
At the test concentration Citrathal Concentrate S TW did inhibit the intrinsic respiration of the inoculum by more than 20% on day 1 of the test. Toxicity towards the inoculum at test concentration can therefore not be excluded.
Executive summary:

The ready biodegradability of Citrathal Concentrate S TW was determined by Manometric Respirometry test according to the OECD Guidelines for Testing of Chemicals, Method No. 301 F. Citrathal Concentrate S TW undergoes 41% biodegradation after 28 days, which was insufficient to consider the substance as readily biodegradable. Since a plateau was not reached, the test was extended. The biodegradation continued reaching a mean level of 56% after 67 days in the test conditions. The individual values for the two replicates were 50 and 62%. The latter meets the pass criterion for inherently and ultimately biodegradable.

At the test concentration Citrathal Concentrate S TW did inhibit the intrinsic respiration of the inoculum by more than 20% on day 1 of the test. Toxicity towards the inoculum at test concentration can therefore not be excluded.

Description of key information

The registered UVCB substance has been tested in an extended ready biodegradation screening test. Based on a result of 62% biodegradation, the substance as a whole is considered as inherently biodegradable for the purpose of classification and labelling. The test results for the whole substance and available data for individual constituents were used to conclude that the substance and underlying constituents are not persistent in the environment.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

The ready biodegradability of Citrathal Concentrate S TW was determined by the Manometric Respirometry test according to the OECD Guidelines for Testing of Chemicals, Method No. 301 F. Due to the complex composition of the test substance, the Biological Oxygen Demand (BOD) was expressed as a percentage of Chemical Oxygen Demand (COD). The latter was determined experimentally according to the DIN 38414 method. Citrathal Concentrate S TW undergoes 41% biodegradation after 28 days in the test conditions, which was insufficient to consider the substance as readily biodegradable. Since a plateau was not reached, the test was extended. The biodegradation continued and reached a mean level of 56% after 67 days in the test conditions. The individual values for the two replicates were 50 and 62%. The latter meets the pass criterion for inherently and ultimately biodegradable.

 

At the test concentration Citrathal Concentrate S TW did inhibit the intrinsic respiration of the inoculum by more than 20% on day 1 of the test. Toxicity towards the inoculum at test concentration can therefore not be excluded.

 

Standard biodegradation tests are not ideally suited to mixtures because they measure ultimate biodegradation as a function of either the CO2 evolved or O2 consumed and as such do not provide information on the biodegradability of individual constituents. Thus for the purpose of the PBT assessment and in order to assign a biodegradation rate to the two assessment entities for environmental risk assessment, available information on the individual constituents was gathered. The data indicates that the C10/C12 oxygenated components in the registered UVCB substance are readily biodegradable while the C7/C10 hydrocarbons are either readily or inherently biodegradable (see separate assessment entity endpoint summaries for details). Thus for the purpose of the environmental exposure and risk assessment the C10/C12 assessment entity has been considered readily biodegradable and, applying a conservative approach, the C7/C10 hydrocarbon group as inherently biodegradable.

 

For the PBT assessment, a result of more than 60% ultimate biodegradability (ThOD, CO2 evolution) in a ready or enhanced ready biodegradability test indicates that the criteria for P are not fulfilled. All known constituents with biodegradation data achieved more than 60% ultimate biodegradability within the standard 28 days of a ready test (i.e. alpha-terpineol, citral, lime dienes, para-cymene, terpinolene) or when the test was prolonged (i.e. Terpinene Alpha). The remaining known constituents are structurally similar to the individually tested substances. They have the same monocyclic “p-menthane” carbon skeleton because the UVCB substance is made from a single raw material, 3,7-dimethylocta-2,6-dienal (Citral), which under the conditions of the production process cyclises to give unsaturated / aromatic hydrocarbons and alcohols (C10 constituents). Due to the presence of ethanol in the process, ethyl ethers of alcohol reaction products may also be formed (C12 constituents). Due to the similar carbon skeleton, these constituents are expected to have reasonably similar biodegradation profiles. Thus all known constituents within the UVCB are considered not persistent for the purposes of the PBT assessment. 

 

In the ECHA guidance on PBT/vPvB assessment (v3, June 2017), the persistency assessment of UVCB substances includes the testing of the whole substance as one approach and states “If the selected test item consists of sufficiently similar structures and is shown to meet the stringent ultimate ready biodegradation test criterion (>60% in 28 days), it can be concluded that the underlying constituents comprising the complex substances are not expected to be persistent (OECD2011). It follows that the same argument may be applied to positive results from an extended screening biodegradation tests. Thus based on the results of replicate 1 for the test performed on Citrathal Concentrate S TW, it is concluded that the registered UVCB substance and its underlying constituents are inherently and ultimately biodegradable and are not persistent in the environment.