3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Biodegradation in water: screening tests

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

adopted 17. Jul. 1992

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 30. May 2008

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

Temperature range was 19.7 – 21.9 °C instead of 20.0 – 24.0 °C. As degradation of the positive control was in the normal range this is considered as uncritical concerning the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

EC Number:

204-729-7

EC Name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Cas Number:

125-20-2

Molecular formula:

C28H30O4

IUPAC Name:

3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide

Details on test material:

- Storage conditions: Room Temperature (20 ± 5 °C)
- Expiry date: 16. Dec. 2019

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant. The chosen plant treats mostly domestic sewage.
- Pretreatment: The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium. It was then aerated until use. The dry matter was determined to contain 5000 mg of suspended solids/L.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Stock solutions:
Solution a
Potassium dihydrogen phosphate (KH2PO4) 8.5 g
Di-potassium hydrogen phosphate (K2HPO4) 21.75 g
Di-sodium hydrogen phosphate dihydrate (Na2HPO4*2H2O) 33.4 g
Ammonium chloride (NH4Cl) 0.5 g
H2O demin. ad 1000 mL
The pH was 7.4.

Solution b
Calcium chloride (CaCl2) 27.5 g
H2O demin. ad 1000 mL

Solution c
Magnesium sulfate heptahydrate (MgSO4*7H2O) 22.5 g
H2O demin. ad 1000 mL

Solution d
Iron(III) chloride hexahydrate (FeCl3*6H2O) 0.25 g
Di-sodium ethylenediamine tetraacetate dihydrate (Na2EDTA*2H2O) 0.4 g
H2O demin. ad 1000 mL

- Test medium:
Solution a 10 mL
Solution b 1 mL
Solution c 1 mL
Solution d 1 mL
H2O demin. ad 1000 mL
The medium was freshly prepared.

- Test temperature: 19.7 – 21.9 °C
- pH: 7.5 at the end of the test
- Aeration of dilution water: yes
- Continuous darkness: yes

SAMPLING
- Sampling frequency: eleven samples were taken within the 28 days
- Sampling method: From each front scrubber flask, 11 samples were taken in order to determine the emitted CO2 (on day 0, 2, 4, 7, 9, 11, 14, 18, 23*, 24 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2.
On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.
*due to error of the carbon analyser on day 23 the measured samples gave wrong results. Therefore, samples were taken on day 24. The results of the samples on day 23 were not used for evaluation.

CONTROL AND BLANK SYSTEM
- Apparatus blanks: 2, containing mineral medium only
- Inoculum blank: 2, containing mineral medium and inoculum
- Abiotic sterile control: 1, containing test item, mineral medium and HgCl2
- Toxicity control: 1, containing test item, positive control, mineral medium and inoculum

Reference substance

Reference substance:

aniline

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

7

Sampling time:

28 d

Details on results:

- The test item 3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide is considered as “not readily biodegradable“.
- The degree of biodegradation reached 7 % after 28 days.
- 10-day-window could not be detected.
- Degradation missed 60 % within 28 days, too. Therefore, the test item is considered as “not ultimately biodegradable within 28 days”, either.
- the abiotic degradation reached 3.6 %.

BOD5 / COD results

Results with reference substance:

Degradation of the positive control surpassed the pass level of 60 % after 8 days.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Degradation behaviour of positive control and toxicity control was normal. Abiotic degradation reached 3.6 %. Both replicates of the test item showed very good correspondence.
If degradation in the toxicity flask is below 25 % after 14 days, the test item can be considered as toxic towards the inoculum. As degradation in the toxicity flask was 38.2 % after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 25.4 mg/L”.
Ready degradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10 %. Therefore, the test item can be considered as “not readily biodegradable”. Degradation missed 60 % within 28 days, too. Therefore, the test item is considered as “not ultimately biodegradable within 28 days”, as well.
Due to error of the carbon analyser on day 23 the measured samples gave wrong results. Therefore, samples were taken on day 24. The results of the samples on day 23 were not used for evaluation.
No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.

Executive summary:

The test item was tested using a concentration of nominally 20 mg organic carbon/L of test item in test medium following OECD 301B and EU-Method C.4-C (under GLP).

Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L). The test was left running for 28 days.

All validity criteria were met. Degradation of the positive control surpassed the pass level of 60 % after 8 days. 

The following data were determined for the test item:

10-day-window: not detected
degradation at the end of the test: 7 %
pass level following guideline: 60 % at the end of 10-day-window for pure substances respective 60 % at the end of the test for mixtures

Therefore, when applying the 10-day-window, the test item is not readily biodegradable following OECD 301B and EU C.4-C respectively. As degradation missed 60% in the course of the test, the substance is considered as not ultimately biodegradable.