Ethyl 1,3-dihydro-1,3-dioxo-2H-isoindole-2-carboxylate

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May 2016 - 18 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate issued on 5 November 2015

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 1,3-dihydro-1,3-dioxo-2H-isoindole-2-carboxylate

EC Number:

245-048-5

EC Name:

Ethyl 1,3-dihydro-1,3-dioxo-2H-isoindole-2-carboxylate

Cas Number:

22509-74-6

Molecular formula:

C11H9NO4

IUPAC Name:

ethyl 1,3-dioxo-2,3-dihydro-1H-isoindole-2-carboxylate

Test material form:

solid

Details on test material:

- Appearance: white solid
- Storage conditions of test material: in refrigerator (2-8°C)

Specific details on test material used for the study:

Carbon content: 60.3 %
Theoretical oxygen demand: 1.825 mg O2/mg (calculated assuming nitrification)
Water solubility: insoluble

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Non-adapted activated sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant; samplimg: on 19 May 2016 (the day before test start)
- Storage conditions and period: sludge was pre-conditioned for 1 day (aerated but not fed) to reduce the amount of O2 consumed by the blank controls
- Preparation of inoculum for exposure: Prior to the test the sludge was washed twice with tap water and once with mineral medium. After centrifugation, the sludge was suspended in test medium at a concentration twice of the final concentration.
- Initial cell/biomass concentration: 30 mg/L dry matter in the final mixture
- Water filtered: no

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

ca. 100 other: mg O2/L

Based on:

ThOD

Initial test substance concentration 2

Initial conc.:

55 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: according to OECD 301F
- Additional substrate: no
- Test temperature: 22 ± 2°C, controlled at ± 1°C, in a thermostat cabinet
- pH: start: 7.2, end: 7.3-7.8
- pH adjusted: yes (to 7.2+/-0.2), only in blank control and proecedure control
- Aeration of dilution water: not during test
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 510 mL glass bottles (tightly closed with manometric BOD measuring devices) containing a total volume of test solution of 200 mL. The bottles are equipped with stirring rods and
butyl rubber quivers which contain 2 pellets of sodium hydroxide each to absorb the produced CO2 from the head space.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration prior to test start + stirring
- Measuring equipment: respirometric activity was measured as pressure change using a manometric BOD measuring device (OxiTop®-C head; WTW Wissenschaftlich-Technische Werkstätten GmbH & Co. KG, DE-82362 Weilheim); DOC was determined with a Shimadzu TOC-Analyzer TOC-L CSH (NPOC mode)
- Test performed in closed vessels: yes

SAMPLING
- Sampling frequency: continuously
- Sampling method: automatic
- Sample storage before analysis: not applicable
- Other: At the end of the test, the pH was measured in all flasks except the abiotic sterile control.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (2 replicates)
- Abiotic sterile control: yes (1 replicate)
- Toxicity control: yes (1 replicate)
- Other: procedural control (2 replicates)

STATISTICAL METHODS: none

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

purity: 99.8%; carbon content: 58.3%; ThOD: 1.665 mg O2/mg

Results and discussion

% Degradation

% Degradation 1

Key result

Parameter:

% degradation (O2 consumption)

Value:

57

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (DOC removal)

Value:

76

Sampling time:

28 d

Details on results:

The biodegradability of the test item based on O2 consumption was calculated to be 57% after 28 days as compared to the theoretical oxygen demand (ThOD).
The biodegradation reached 53% at the end of the 10-d window (i.e. within 10 days after attainment of 10% degradation).
Biodegradation of the test item was observed after a lag-phase of about 5 days.

The respective concentrations of organic carbon at the beginning (theoretical value based on the test item’s carbon content and the test concentration, as mg TOC/L) and at the end of the test (mean measured value of the two replicates, as mg DOC/L) were, respectively:
33.2 and 8.82 for the test units
<0.5 and 1.27 for the blank control
35.0 and 1.04 for the procedure control.
The total elimination based on dissolved organic carbon (DOC) measurements reached 76% for the test item and 101% for sodium benzoate, respectively. This data confirm that only partial degradation of the test item occurred.

The procedure control sodium benzoate reached 86% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.

At the applied initial test concentration of 55 mg/l the test item was not judged to have any inhibitory effect on the microbial population, since the biodegradation of the mixture (test item + reference item sodium benzoate) exceeded 25% within 14 days.

The test time was not abiotically degraded (by processes using O2) during the whole test period of 28 days in the absence of microorganisms as confirmed by the lack of oxygen consumption in the abiotic sterile control.

BOD5 / COD results

Results with reference substance:

86% biodegradation after 14 days

Any other information on results incl. tables

The O2 uptake values and degradation data are summarized in the tables attached under "Attached background material".

Validity criteria

1. The difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the 10-d window, or at the end of the test, as appropriate, must be less than 20% - actual: 6% at the end of the 10 -d window (day 20) and 8% at test end (day 28)

2. The percentage of degradation of the reference item must reach the pass level of 60% by day 14 - actual: 86% by day 14

3. In the toxicity control, the degradation of the mixture test item + reference item must reach the pass level of 25% by day 14; otherwise, the test item can be assumed to be inhibitory and the test should be repeated using a lower concentration of test item - actual: 67% by day 14

4. The O2 uptake in the blank control should normally be 20-30 mg O2/L and should not be greater than 60 mg O2/L in 28 days. - actual: 19.6 mg O2/L

5. The pH value should be in the range of 6-8.5. - at test end: pH blanks - 7.3, pH test item solution - 7.3 and 7.8; pH reference item solution - 7.5 and 7.6; pH toxicity control - 7.5

All validity criteria were fulfilled.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see "Any other information on results..."

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

In the Manometric Respirometry Test according to OECD 301F the test item was found to biodegrade (based on O2 consumption) for 57% after 28 days of incubation and, therefore, cannot be termed as readily biodegradable.

Executive summary:

In a test perfomed according to OECD 301F (Manometric Respirometry Test) and GLP, the test item did not reach the pass level of 60% for ready biodegradability, neither within the 10 -d window nor after 28 days of incubation. It can therefore not be termed as readily biodegradable.