2,3-dihydro-2,2,6-trimethylbenzaldehyde

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:

supporting study

Study period:

March 14, 2014 to July 29, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

yes

Remarks:

Extended beyond the standard 28 days.

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Duration of test (contact time):

70 d

Initial conc.:

30 mg/L

Based on:

test mat.

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

61

Sampling time:

70 d

Details on results:

As < 60% biodegradation was observed after 28days, the ready biodegradability test was prolonged in order to assess the inherent biodegradability of the substance. Biodegradation began around day 30 and gradually increased to a level of 61% after 70 days. From the timeplot the > 60% biodegradation was achieved from day 66 onwards.

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.
Oxygen uptake curves can be found in Appendix 1 of the attached study report. Calculated % biodegradation curves and detailed results are reported in Appendixes 2 to 4 of the attached study report.
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 on day 28 of the test, key date for the interpretation of the results. Therefore, the test is considered valid.

Validity criteria fulfilled:

yes

Interpretation of results:

other: Not readily biodegradable but Inherently and ultimately biodegradable

Conclusions:

The biodegradability of SAFRANAL P was determined by the Manometric Respiratory Test according to OECD Guidelines No 301F. Under the conditions of the test, no biodegradation was observed after the standard 28 day test period. Thus, SAFRANAL P should be regarded as not readily biodegradable according to this test.

As less than 60% biodegradation was observed after 28 days, the ready biodegradability test was prolonged in order to assess the inherent biodegradability of the substance. Biodegradation began around day 30 and gradually increased to a level of > 60%, 61% after 70 days. From the timeplot the > 60% pass criterion was achieved from day 66 onwards. Based on these results SAFRANAL P should be regarded as inherently and ultimately biodegradable.

SAFRANAL P did not significantly 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 of 30mg/L.

Executive summary:

The ready biodegradability test was carried out according to OECD TG 301F guideline under GLP.

Safranal P undergoes no biodegradation after 28 days (61% after 70 days) in the test conditions.

Safranal P did not significantly 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.

Thus, Safranal P should be regarded as not readily biodegradable according to this test. However, based on these results Safranal P should be regarded as inherently and ultimately biodegradable.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From December 21, 2018 to February 22, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Details on inoculum:

Source: The source of test organisms was secondary effluent freshly obtained from a municipal sewage treatment plant: receiving predominantly domestic sewage from the Sewage Treatment plant, Chandana Complex, Tumkur-572103, India
Treatment: Secondary effluent was filtered through a coarse filter paper; the first 200 mL was discarded. The filtrate was kept aerated upto inoculation.
Inoculation: A volume of 5 mL filtrate of Secondary effluent was added per L of final volume (i.e. 1.5mL of filtrate was added for 300 mL of final volume).

Duration of test (contact time):

28 d

Initial conc.:

8 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Reference substance:

acetic acid, sodium salt

Key result

Parameter:

% degradation (O2 consumption)

Value:

7.7

Sampling time:

28 d

Parameter:

ThOD

Value:

2.77 g O2/g test mat.

Results with reference substance:

ThOD: 0.78g of O2/g reference substance

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item (Safranal) is non-readily biodegradable under the conditions of the closed bottle test according to the OECD guideline 301 D.

Executive summary:

The ready biodegradability of the Safranal was determined using closed bottle test as per OECD guideline No. 301 D, 1992.

Test item was a pale yellow to yellow colored liquid with a purity of 95.4%. The ThOD of test item (Safranal) was calculated and found to be 2.77 mg of O₂/mg. For the calculation of the ThOD, the molecular formula and molecular mass of the test item was used. The ThOD of sodium acetate (reference substance) was calculated and found to be 0.78mg of O₂/mg.

During the study, the determination of oxygen concentration were performed at the start of the experiment (day 0) and on days 7, 10, 14, 21 and 28.

The test item reached a maximum biodegradability of 7.70% based on ThOD within 28 days of biodegradability which is not exceeding 60%.

The difference of duplicate biodegradation values on day 28 expressed as mg O₂/L were less than 20% (3.52%).

After 28 days oxygen depletion in the inoculum blank obtained was 0.88mg O₂/L which is below 1.5mg O₂/L.

The residual concentrations of oxygen in the test bottles were found to be > 0.5mg/L at all durations.

The reference substance (sodium acetate) attained a maximum 62.29% biodegradation based on ThOD (which is greater than 60% degradation) within the 14 days period.

 

Conclusion

The test item (Safranal) is non-readily biodegradable under the conditions of the closed bottle test according to OECD criteria.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 26, 2020 to January 28, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

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.

Duration of test (contact time):

60 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Reference substance:

other: Sodium benzoate

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.

Key result

Parameter:

% degradation (O2 consumption)

Value:

30

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Value:

73

Sampling time:

60 d

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 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.

From the time plot the > 60% biodegradation was achieved from day 42 onwards.

Validity criteria fulfilled:

yes

Interpretation of results:

other: Not readily biodegradable but Inherently biodegradable

Conclusions:

The Ready Biodegradability of Safranal P was determined by the Manometric Respirometry Test.
Safranal P undergoes 30% biodegradation after 28 days and 73% after 60 days in the test conditions. The 10-day window criterion is therefore not fulfilled (10% on day 21 and 39% on day 31 ).
Safranal P 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.
Thus, Safranal P should be regarded as not readily biodegradable but inherently biodegradable according to this test.

Executive summary:

The Ready Biodegradability of Safranal P was carried out according to OECD TG 301 F Guideline (by the Manometric Respirometry Test), under GLP.

Safranal P undergoes 30% biodegradation after 28 days and 73% after 60 days in the test conditions. The 10-day window criterion is therefore not fulfilled (10% on day 21 and 39% on day 31 ).

Safranal P 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.

Thus, Safranal P should be regarded as not readily biodegradable but inherently biodegradable according to this test.

Description of key information

Safranal is not readily biodegradable based on the results of three valid OECD 301 screening studies. However, it is considered to be inherently and ultimately biodegradable, and not persistent based on > 60% biodegradation in two prolonged OECD 301F ready tests.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, fulfilling specific criteria

Type of water:

freshwater

Additional information

Three studies to assess the ready biodegradability of the registered substance are available; two prolonged 301F studies (Test Item Safranal P, Mendoza 2021, key study and Kreutzer 2014, supporting study) and a 301D study (Test Item Safranal, Sundari, 2019, supporting study). The percentage biodegradation after 28 days was 30, 9 and 7.7% respectively. Thus, 2,6,6 -trimethylcyclohexa-1,3 -diencarbaldehyde is regarded as not readily biodegradable according to all three tests.  

The registered substance 2,6,6-trimethylcyclohexa-1,3-dienecarbaldehyde is a mono-constituent substance (EC 204-133-7, CAS 116-26-7). Two commercial qualities are covered by the REACH lead registrant, Safranal and Safranal P. The only difference between the two qualities is the amount of the additive, alpha-tocopherol (EC 233-466-0, CAS 10191-41-0), that is intentionally added to stabilise the substance. The level of this additive is 9-14% in Safranal and 1-2% in Safranal P.

Safranal P was used in the biodegradation studies performed by the lead registrant (Kreutzer 2014 and Mendoza 2021) because of the lower alpha-tocopherol level and hence higher purity of mono-constituent substance. Both of these studies were performed according to OECD guideline 301F, under GLP. Since less than 60% biodegradation was observed after 28 days, the studies were prolonged in order to assess the ultimate biodegradability of the substance. Both studies showed a lag phase with biodegradation beginning at around 16 days (Mendoza 2021) or 30 days (Kreutzer 2014). Biodegradation then continued to gradually increase with the pass level of 60% attained after 42 and 66 days respectively. The final biodegradation levels achieved were 73% (60d, Mendoza 2021) and 61% (70d, Kreutzer 2014). Based on these results SAFRANAL P is regarded as inherently and ultimately biodegradable. Furthermore, given that a level of >60% biodegradation was achieved, the substance is not expected to be persistent in the environment for the purpose of the PBT assessment.

Both OECD 301F tests were performed at a test concentration of 30mg/L. There was no evidence in either test that SAFRANAL P significantly inhibited the intrinsic respiration of the inoculum. Therefore SAFRANAL P is considered to be non-toxic to STP inoculum at the test concentration of 30mg/L.

The Mendoza 2021 study is considered the key study because it exhibited the shortest lag phase and attained the highest level of biodegradation at test end. The other two studies are considered as supporting. This is line with ECHA guidance R.7.9.4 where it is recognised that due to the stringent conditions of ready biodegradability tests, positive test results should supersede negative test results. It follows, therefore, that any test giving more positive results (e.g. higher degradation levels, shorter lag phase) should take precedence.