Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis([1,1'-biphenyl]-4-yl)-2,5-dihydro-

Biodegradation in water and sediment: simulation tests

Administrative data

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

additional information on environmental fate and behaviour

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: OECD 318

At any of the time points mentioned in the TG-318, the influence of Ca is critical. Regardless of pH, the pigment is least stable in 10 mM Ca, representing high water hardness.
After 6h, the samples showed high dispersion stability in 0 mM Ca at pH = 9 and pH = 7 and intermediate stability in 0 mM Ca and pH = 4. For the samples at all other conditions the stability was low.
After 24 hours the stability in 0 mM Ca at pH = 9 remained high and at pH = 7 and pH = 4 was intermediate. For the samples at all other conditions the stability was low.

 

Full results of the dispersion stability in the presence of NOM
	Ca(NO3)2	Stability after 6h	Standard deviation	Stability after 15h	Standard deviation	Stability after 24h	Standard deviation
	[mM]	[%]	[%]	[%]	[%]	[%]	[%]
pH 4	0	87.7	0.9	82.1	0.2	77.9	0.3
pH 4	1	13.2	1.5	5.7	0.1	4.1	0.7
pH 4	10	26.3	1.0	11.7	0.2	7.7	0.4
.
pH 7	0	96.0	0.3	91.3	0.4	85.0	3.0
pH 7	1	13.2	2.1	5.7	0.6	4.2	0.5
pH 7	10	15.4	1.0	6.4	0.5	4.1	0.5
.
pH 9	0	96.1	0.9	93.9	2.2	91.7	2.2
pH 9	1	30.3	2.2	12.4	1.2	8.5	0.7
pH 9	10	14.7	0.7	7.0	0.6	5.4	0.4

 

To rationalize the observed dispersion stability, we finally checked the particle size distribution directly in the environmental medium. We applied the NanoDefine method of Analytical Ultracentrifugation (SOP AUC-RI, published by 3). The centrifugation parameters are given in the methods section.
As required by TG318, paragraph 31, the tested nanomaterial was pre-wetted in ultrapure water and left in the form of wet-paste for 24 h. The TG318 requires this step “to insure the proper interaction of nanomaterial surface with ultrapure water.” We visually observed incomplete wetting, and so any ensuing measurement would have been incorrect. In accord with the NanoGenoTox dispersion protocol, a drop of ethanol was added, successfully transferred the powder into a paste, which was then further diluted as specified in the TG318
The observed size distributions confirm the moderate agglomeration at 1 mM Ca, pH7, with NOM (Figure 4). If the particles would have been significantly dissolved, no size distribution would be observable at all by this method, which relies on the detection of the movement of particles during centrifugal separation.
Additionally, the centrifugation methods include a determination of the remaining absorption after centrifugation, fully consistent with the conventional determination of the dissolved fraction after centrifugation as recommended by the TG-318. The remaining absorption was measured at ca. 0.08. This is a fraction of approximately 6% of the initial absorption. Considering the LOD, between 0% and 6% of the sample may have been dissolved.
All evidence combined, the results after centrifugation confirm that at least 94% of the observed dispersion stability has to be attributed to the particles, not to dissolution.

 

 

Executive summary:

At any of the time points mentioned in the TG-318, the influence of Ca is critical. Regardless of pH, the pigment is least stable in 10 mM Ca, representing high water hardness.
After 6h, the samples showed high dispersion stability in 0 mM Ca at pH = 9 and pH = 7 and intermediate stability in 0 mM Ca and pH = 4. For the samples at all other conditions the stability was low.
After 24 hours the stability in 0 mM Ca at pH = 9 remained high and at pH = 7 and pH = 4 was intermediate. For the samples at all other conditions the stability was low.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

The substance is completely within the parametric domain and the metabolic domain of the model. However, the substance is not in the structural domain, due to 85.71% unknown fragments in the training set.

Justification for type of information:

1. SOFTWARE
OASIS Catalogic v5.15.2

2. MODEL (incl. version number)
CATALOGIC 301C v12.17

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
See section 'Test Material'.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF.

5. APPLICABILITY DOMAIN
See attached QPRF.

6. ADEQUACY OF THE RESULT
- The model is scientifically valid (see attached QMRF).
- The model estimates the biodegradability of a substance Screening information on the ready biodegradability is required for substances manufactured or imported in quantities of 1 t/y or more. Depending on the results, further information may be required for substances manufactured or imported in quantities of 100 t/y or more (simulation testing on ultimate degradation in surface water/soil/sediment). Column 2 of REACH Annex VII provides exemptions for conducting the study. It does not need to be conducted if the substance is inorganic. According to column 2 of REACH Annex IX, testing is not required if the substance is highly insoluble in water, or the substance is readily biodegradable.
- See attached QPRF for reliability assessment.

Principles of method if other than guideline:

Estimation of ready biodegradation in water using CATALOGIC v5.15.2 BOD 28 days MITI (OECD 301C) v12.17

GLP compliance:

no

Oxygen conditions:

aerobic

Inoculum or test system:

other: model calculation

Duration of test (contact time):

28 d

Parameter followed for biodegradation estimation:

O2 consumption

Parameter:

% degradation (O2 consumption)

Value:

26

Sampling time:

28 d

Remarks on result:

other: The substance is not within the applicability domain of the model due to 85.71% unknown fragments in the training set.

-Concomitant predictions:

Not ready degradable

Primary Half Life = < 1 days

Ultimae Half Life = 2m 5d

 

- Predicted value (model result): O2 -consumption (BOD) = 0.26 ± 4.03E-3

 

Metabolite predition:

The model Catalogic 301 C v11.15 (OASIS Catalogic v5.13.1.156) calculated 40 metabolites (see document "2022-06-30_88949-33-1 PR 264_Catalogic Metabolites" attached under "attached background material") identifying 20 metabolites as relevant degradation products in terms of PBT/vPvB assessment, with an estimated quantity of ≥ 0.1%.

All metabolites identified as relevant degradation products were calculated to be not readily biodegradable. Individual biodegradation of these metabolites range from 11 to 29 % after 28 days (based on BOD). All metabolites, exept one (metabolite # 1.2) are not bioaccumulative according PBT criteria due to the calculated logKow below 4.5. For the metabolite #1.2 a logKow of 4.7 has been calculated. This value is slightly above the threshold of 4.5 given by the PBT criteria. Nevertheless, this metabolite has a very similar structure compared to the parent compound. The 2,5-dihydro-pyrrolo(3,4-c)pyrrole-1,4-dione core is opened. However, structure, moelcular weight and water solubilty are very similar compared to the parent compound. Therefore, since the parent compound is not bioaccumulative, it can be assumed with high probability that the metabolite #1.2 is not bioaccumulative according PBT criteria either.

Interpretation of results:

not readily biodegradable

Remarks:

according to OECD criteria

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf / Switzerland) treating predominantly domestic wastewater
- Preparation of inoculum for exposure: The sludge was washed three times with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of two days prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was diluted with test water to a concentration of about 1 g dry material per liter. Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

20 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST WATER
The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:
1) KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HP04x2H20 33.40 g/L
NH4CI 0.50 g/L
The pH of this solution was 7.4.
2) MgS04x7H20 22.50 g/L
3) CaCl2x2H20 36.40 g/L
4) FeCl3 X 6H2O 0.25 g/L, stabilized with one drop of concentrated HCI per liter

To obtain the final test water, 10 mL of stock solution 1) and 1 mL each of stock solutions 2) - 4) were added to about 800 mL purified water and made up to 1000 mL with purified water. The pH of the final test water was adjusted from 7.9 to 7.4 with a diluted hydrochloric acid solution.

PREPARATION OF TEST FLASKS
One day before test start (Day -1), between 2400 and 3000 mL of untreated test medium was filled into 5-liter flasks (amber glass). To each flask, 90 mL activated sludge inoculum was added.
The test media were aerated overnight with C02-free air to purge the system of carbon dioxide.
On the following day (Day 0), defined amounts of the test item were directly added to the test flasks. No emulsifiers or solvents were used.
The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 770 mg sodium benzoate per 100 mL test water (purged with C02-free air) was prepared. From this, 10 mL aliquots were added to the corresponding test flasks.
The test flasks were made up to a volume of three liters with test water (purged with C02-free air). Two absorber flasks, the first one containing 300 mL 0.05 M NaOH and the second one containing 200 mL 0.05 M NaOH, were connected in series to the exit air line of each test flask.

TEST CONDITIONS
Test vessels: 5-liter all-glass amber bottles.
Identification: The test vessels were labeled with the necessary information to ensure unmistakable identification.
Test duration: 28 days
Light conditions: The test vessels were incubated in a dark room.
Test temperature:
20 - 22 °C. The inoculated flasks were incubated in a temperature controlled
room. The temperature was checked on each sampling date in a separate flask with purified water. Additionally, the room temperature was continuously recorded.
C02-free air: Air was led through a bottle containing about 750 mL of a 2 M NaOH
solution to trap CO2. The C02-free air was passed through the test solutions at a rate conesponding to about 30-100 mL/min.
pH: Prior to test start (Day 0), the pH was measured in each test flask after the addition of test and/or reference item and, if necessary, adjusted to 7.4 ± 0.2 with a diluted sodium hydroxide solution. At the end of incubation (Exposure Day 28), the pH was measured again in each test flask.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

11

Sampling time:

28 d

Results with reference substance:

The percent biodegradation of the reference item was calculated based on a total carbon content (TOC) of 0.58 mg C/mg sodium benzoate.
In the procedure controls, average biodegradation of the reference item was 89% by Exposure Day 14, thus confirming suitability of the activated sludge (>60%) degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 99%.

Biodegradation of the Test Item

The percent biodegradation of the test item was calculated based on a total carbon content (TOC) of 0.82 mg C/mg test item.
The CO2 formation of the test item in the test media was only slightly higher than in the inoculum controls. Degradation amounted to maximum 11% at
test end.

Biodegradation of the Toxicity Control

The percent biodegradation in the toxicity control, containing both the test item and the reference item, was calculated based on the sum of the total carbon content (TOC) of the test item and the reference item.
CO2 formation in the toxicity control showed a similar course over the 28-day exposure period as the two procedure controls, containing only the reference item. Within 14 days of exposure, biodegradation reached 33%. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 20 mg/L because biodegradation in the toxicity control was >25% within 14 days of incubation.

 

Driving Off CO2
No significant difference was found between the absolute amounts of IC measured on Exposure Day 28 and the absolute amounts of IC measured after acidification on Day 29.
Consequently, no residual CO2 was present in the test solutions or suspensions at the end of the test.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test substance is not readily biodegradable according to OECD criteria.

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

413-920-6

EC Name:

-

Cas Number:

88949-33-1

Molecular formula:

C30 H20 N2 O2

IUPAC Name:

3,6-bis({[1,1'-biphenyl]-4-yl})-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

Test material form:

solid: nanoform

Details on test material:

- State of aggregation: solid
- Particle size distribution (TEM): 27.8 nm (D50)
- Mass median aerodynamic diameter (MMAD): not specified
- Geometric standard deviation (GSD): not specified
- Shape of particles: spherical
- Surface area of particles (BET): 100 m^2/g
- Crystal structure: crystalline
- Coating: no
- Surface properties: not applicable
- Density: 1390 kg/m^2
- Moisture content: < 0.3 %
- Residual solvent: < 50 ppm 2-methylbutan-2-ol (tert-amylalcohol), CAS 75-85-4
- Activation: not applicable
- Stabilisation: not applicable