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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study according to 87/302/EEC (1987) without analytical monitoring

Qualifier:

according to guideline

Guideline:

other: 87/302/EEC, 1987

Deviations:

yes

Remarks:

, in order to investigate the potential inhibitory effect of a substance with a water solubility far below the available analytical methods, the test was conducted with a saturated aqueous extract.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In order to prepare the stock solution 100 mg of the test substance were suspended in 1000 mL of testwater which was continously agitated during 48h prior filtration through a 0.22 µm glassfiber filter. The nominal test concentrations were achieved by using the stock solution as the highest concentration and by mixing the stock solution and test water in a 1:1 ratio. The stock solution appeared to be colorless.
- Controls: Blank water

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Source (laboratory, culture collection): CIBA-GEIGY Ltd., testing facility
- Preculture: 3 days under test conditions

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 25 °C

pH:

Values from 7.2 to 8.4

Nominal and measured concentrations:

Nominal: Stock solution and stock solution with water mixture (ratio 1:1)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml Erlenmeyer flasks, stoppered with aluminium caps, on Lab-shaker, 50 ml test solution per flask
- Initial cells density: 11600 cells/ml
- Control end cells density: 181.9E4 cells/ml (mean density)
- No. of vessels per concentration (replicates): 3
- No. of control vessels: 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Composition according to guideline
- Intervals of water quality measurement: Temperature was continuously measured and maintained at 23 ± 1 °C. pH was measured at test start and 72 hours of exposure

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination, cold white fluorescent light, 120 µE/m²sec ± 20 % (ca. 8 klux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities were measured at 24, 48 and 72 hours of exposure on a "TOA" cell counter.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

growth rate

Remarks on result:

other: EC 50 (0-72 h) > saturated solution

Remarks:

no effects within the range of solubility

Key result

Duration:

72 h

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

growth rate

Remarks on result:

other: NOEC (0-72 h) > saturated solution

Remarks:

no effects within the range of solubility

Details on results:

- Exponential growth in the control (for algal test): yes

Nominal concentration of treatment [mg/L]	Inhibition compared to control [%]
0.5	1.7
1.0	0

0.5: saturated solution diluted 1:1 with test water

1.0: saturated solution in test water

Validity criteria fulfilled:

yes

Conclusions:

In an algae toxicity study, the substance did not cause any effects (growth and cell density) using a saturated solution.

Executive summary:

The purpose of this study was to evaluate the algae toxicity of the test substance on Scebedesmus subspicatus similar to EU Test Method C.3.

Algae were exposed to a saturated stock solution as the highest concentration and to a mixture of the stock solution and test water in a 1:1 ratio for 72 hours. A concurrent negative control groups were also tested.

The algae's cell density and growth were measured at 24, 48 and 72 hours.

All validity criteria were met. No toxicity of the test substance was observed.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study according to 87/302/EEC (1987) without analytical monitoring

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.

3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms form non-dispersible aggregates in aqueous milieu, read-across of aquatic toxicity studies from the source to the target form is scientifically justified.

4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: 87/302/EEC, 1987

Deviations:

yes

Remarks:

, in order to investigate the potential inhibitory effect of a substance with a water solubility far below the available analytical methods, the test was conducted with a saturated aqueous extract.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In order to prepare the stock solution 100 mg of the test substance were suspended in 1000 mL of testwater which was continously agitated during 48h prior filtration through a 0.22 µm glassfiber filter. The nominal test concentrations were achieved by using the stock solution as the highest concentration and by mixing the stock solution and test water in a 1:1 ratio. The stock solution appeared to be colorless.
- Controls: Blank water

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Source (laboratory, culture collection): CIBA-GEIGY Ltd., testing facility
- Preculture: 3 days under test conditions

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 25 °C

pH:

Values from 7.2 to 8.4

Nominal and measured concentrations:

Nominal: Stock solution and stock solution with water mixture (ratio 1:1)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml Erlenmeyer flasks, stoppered with aluminium caps, on Lab-shaker, 50 ml test solution per flask
- Initial cells density: 11600 cells/ml
- Control end cells density: 181.9E4 cells/ml (mean density)
- No. of vessels per concentration (replicates): 3
- No. of control vessels: 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Composition according to guideline
- Intervals of water quality measurement: Temperature was continuously measured and maintained at 23 ± 1 °C. pH was measured at test start and 72 hours of exposure

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination, cold white fluorescent light, 120 µE/m²sec ± 20 % (ca. 8 klux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities were measured at 24, 48 and 72 hours of exposure on a "TOA" cell counter.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

growth rate

Remarks on result:

other: EC 50 (0-72 h) > saturated solution

Remarks:

no effects within the range of solubility

Key result

Duration:

72 h

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

other: saturated solution

Basis for effect:

growth rate

Remarks on result:

other: NOEC (0-72 h) > saturated solution

Remarks:

no effects within the range of solubility

Details on results:

- Exponential growth in the control (for algal test): yes

Nominal concentration of treatment [mg/L]	Inhibition compared to control [%]
0.5	1.7
1.0	0

0.5: saturated solution diluted 1:1 with test water

1.0: saturated solution in test water

Validity criteria fulfilled:

yes

Conclusions:

In an algae toxicity study, the substance did not cause any effects (growth and cell density) using a saturated solution.

Executive summary:

The purpose of this study was to evaluate the algae toxicity of the test substance on Scebedesmus subspicatus similar to EU Test Method C.3.

Algae were exposed to a saturated stock solution as the highest concentration and to a mixture of the stock solution and test water in a 1:1 ratio for 72 hours. A concurrent negative control groups were also tested.

The algae's cell density and growth were measured at 24, 48 and 72 hours.

All validity criteria were met. No toxicity of the test substance was observed.

Description of key information

In an algae toxicity study, the substance did not cause any effects (growth and cell density) using a saturated solution.

Additional information

As the substance did not induce any effects at its maximum water solubility, no key value is indicated.