5-(3-Phenylpropioloyl)-1,3-isobenzofurandione

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2019 - 24 October 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: NEXAMITE® A56
Chemical name: Phenylacetylene modified trimellitic anhydride
CAS number: 1329658-14-1
Batch/Lot number: NEX-X61-A20
Description: Light yellow powder
Molecular formula: C17H8O4
Molecular Weight: 276.25 g/mol
Purity: >98%
Expiry date: 23 October 2019
Storage conditions: Controlled room temperature (15-25oC, ≤70% relative humidity), protected from humidity (tight closed container) under inert gas
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety.

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

The inoculum: Secondary effluent, microorganisms from a domestic waste water treatment plant.

Origin: The secondary effluent was supplied from the sewage plant for domestic sewage in Veszprém county, Hungary.
Conditioning: The secondary effluent used for this study was allowed to settle approximately for an hour, then the decanted effluent was aerated until use (not later than an hour).

Duration of test (contact time):

28 d

Initial conc.:

3 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST UNITS

Type and Size: BOD bottles (300 mL) with special neck and glass stoppers.
Identification: Each test flask was uniquely identified with study code, test group, days of measurement and replicate number.

TEST CONDITIONS
The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22  2 C according to guideline. The test flasks were placed into an incubator and kept at 21.0 - 23.0C, in the dark. The temperature was measured on weekdays during the experiment.
The oxygen concentration of test water was 8.8 mg/L at the start of the test.
The pH value of the test water was checked prior start of the experiment. The pH of the test water was 7.27.

The test conditions were measured with suitable instruments and documented in the raw data.

PREPARATION OF THE TEST SOLUTIONS

The respective amount of Test Item was weighed in directly to reach the required test item concentration of 3.0 mg/L.
The chosen test item concentration was based on the theoretical oxygen demand (ThOD) of 1.97 mg O2/mg test item (calculated according to equation given in the guidelines) and on the performed 14-d preliminary test. During the preparation of the test solutions they were mixed by mechanical stirring using ultrasonic bath for 10 minutes.

The components were applied in the amounts/volumes in the test flasks:

1.) Test Item (flasks 1a and 1b)

Based on the theoretical oxygen demand (ThOD) of 1.97 mg O2/mg test item,
(10.2 mg of Test Item) was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 3.0 mg/L test item, with a ThOD of about 5.910 mg O2/L).

2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)

Based on the theoretical oxygen demand (ThOD) of Sodium benzoate (1.67 mg
O2 per mg; details on calculation are given in the guidelines), stock solution* corresponding to 12.24 mg of Sodium benzoate was mixed into 3.40 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThOD of about 6.012 mg O2/L).

* The concentration of the stock solution was: 360 mg/L.

3.) Inoculum Control (flasks 3a and 3b)

Only filtered inoculum was added to 3.40 litres of aqueous test medium.

4.) Toxicity Control (flasks 4a and 4b)

The Test Item (10.2 mg) and the reference item stock solution* (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 3.0 mg/L test item (ThOD of 5.910 mg O2/L) and 3.6 mg/L reference item (ThOD of 6.012 mg O2/L).

* The concentration of the reference item stock solution was: 360 mg/L.

Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.

Preparation of Test Flasks

Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.
Test item (flasks 1a and 1b) and procedure control (flasks 4a and 4b) test solutions were agitated on each working day during the experiment.

The Test Bottles

The number of test bottles was the follow:
 10 bottles containing the test item and inoculum
 10 bottles containing the reference item and inoculum (procedure control)
 10 bottles containing only inoculum (inoculum control)
 10 bottles containing the test item, reference item and inoculum (toxicity control)

The reference item and inoculum (procedure control) were also part of a concurrent study (Citoxlab study code: 19/176-322AN) performed in the same experimental period using the same batch of chemicals and solutions. Raw data sheets are clearly associate data with the appropriate study number. Copies of all raw data of positive and negative controls run in parallel were placed in the study file.

Measurement of Oxygen
The incubation period of the closed bottle test was 28 days.
The oxygen concentrations were measured with an oxygen meter with a stirring O2 electrode. Oxygen measurements were performed in duplicate on days 0, 7, 14, 21 and 28.

Measurement of Temperature
Temperature was measured continuously and registered on weekdays.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Not applicable

Test performance:

CONDITIONS FOR THE VALIDITY OF THE TEST

- Oxygen depletion in the inoculum control did not exceed 1.5 mg O2/L after 28 days.
- The residual oxygen concentration in the test flasks did not drop below 0.5 mg O2/L at any time.
- The difference of extremes of replicate values of the removal of the test substance at the plateau, (at the end of the test or at the end of the 10 or 14-d window) was not greater than 20 %.
- The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) within 14 days.
- The percentage degradation in the toxicity control containing both the test item and the reference item reached the level for ready biodegradability (> 25 %) by exposure day 14.

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test item

Value:

>= 0.49 - <= 0.5

Sampling time:

7 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test item

Value:

>= 1.2 - <= 1.2

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test item

Value:

>= 1.15 - <= 1.25

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test item

Value:

>= 1.3 - <= 1.4

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Reference item

Value:

>= 4.24 - <= 4.48

Sampling time:

7 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Reference item

Value:

>= 4.6 - <= 4.7

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Reference item

Value:

>= 4.85 - <= 4.85

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Reference item

Value:

>= 5 - <= 5.1

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test item Control

Value:

3.98

Sampling time:

7 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test item Control

Value:

4.8

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test item Control

Value:

4.95

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Test item Control

Value:

5.2

Sampling time:

28 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Reference item control

Value:

4.1

Sampling time:

7 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Reference item control

Value:

5

Sampling time:

14 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Reference item control

Value:

5.45

Sampling time:

21 d

Key result

Parameter:

% degradation (O2 consumption)

Remarks:

Reference item control

Value:

5.5

Sampling time:

28 d

Key result

Parameter:

BOD5

Value:

>= 0.16 - <= 0.17 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Test item, 7 days

Key result

Parameter:

BOD5

Value:

>= 0.4 - <= 0.4 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Test item, 14 days

Key result

Parameter:

BOD5

Value:

>= 0.38 - <= 0.42 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Test item, 21 days

Key result

Parameter:

BOD5

Value:

>= 0.43 - <= 0.47 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Test item, 28 days

Key result

Parameter:

BOD5

Value:

>= 1.18 - <= 1.24 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Reference item, 7 days

Key result

Parameter:

BOD5

Value:

>= 1.28 - <= 1.31 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Reference item, 14 days

Key result

Parameter:

BOD5

Value:

>= 1.35 - <= 1.35 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Reference item, 21 days

Key result

Parameter:

BOD5

Value:

1.39 - 1.42 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Reference item, 28 days

Key result

Parameter:

BOD5

Value:

0.6 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Test item control, 7 days

Key result

Parameter:

BOD5

Value:

0.73 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Test item control, 14 days

Key result

Parameter:

BOD5

Value:

0.75 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Test item control, 21 days

Key result

Parameter:

BOD5

Value:

0.79 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Test item control, 28 days

Key result

Parameter:

BOD5

Value:

0.62 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Reference item control, 7 days

Key result

Parameter:

BOD5

Value:

0.76 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Reference item control, 14 days

Key result

Parameter:

BOD5

Value:

0.83 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Reference item control, 21 days

Key result

Parameter:

BOD5

Value:

0.83 mg O2/g test mat.

Remarks on result:

other:

Remarks:

Reference item control, 28 days

Results with reference substance:

The reference item Sodium benzoate was sufficiently degraded to a mean of 77.5 % after 14 days, and to a mean of 84.2 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the test conditions the percentage biodegradation of NEXAMITE® A56 reached a mean of 22.9 % after 28 days based on the ThOD of the test item. According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore, the test item is considered not readily biodegradable.

Executive summary:

The testitem (NEXAMITE® A56)wasinvestigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

 

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

Under the test conditions the percentage biodegradation of NEXAMITE® A56reached a mean of 22.9 % after 28 days based on the ThOD of the test item.According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD. Therefore, the test item is considered not readily biodegradable.

 

The reference item Sodium benzoate was sufficiently degraded to a mean of 77.5 % after 14 days, and to a mean of 84.2 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

 

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 40.8 % biodegradation was noted within 14 days and 44.6 % biodegradation after 28 days of incubation.

 

According to the test guidelines the test item can be assumed to be not inhibitory at the applied concentration level of 3.0 mg/Lon thesecondary effluent microorganismsbecause degradation was >25 % within 14 days.

Description of key information

The test item (NEXAMITE® A56)was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

 

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

 

Under the test conditions the percentage biodegradation ofNEXAMITE® A56reached a mean of 22.9 % after 28 days based on the ThOD of the test item.According to the test guidelines the pass level for ready biodegradability is removal of 60 % ThOD.Therefore, the test item is considered not readily biodegradable.

 

The reference item Sodium benzoate was sufficiently degraded to a mean of 77.5 % after 14 days, and to a mean of 84.2 % after 28 days of incubation, based on ThOD, thus confirming the suitability of the used inoculum.

 

In the toxicity control containing both, the test item and the reference item Sodium benzoate, a mean of 40.8 % biodegradation was noted within 14 days and 44.6 % biodegradation after 28 days of incubation.

 

According to the test guidelinesthe test item can be assumed to be not inhibitory at the applied concentration level of3.0 mg/Lon thesecondary effluentmicroorganismsbecause degradation was >25 % within 14 days.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater