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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

adult fish: sub(lethal) effects

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 OECD Guideline 204 without analytical monitoring

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

Version / remarks:

Version from 1984

Deviations:

yes

Remarks:

The test concentration was test water saturated with test substance; the saturation was achieved using a flow through system; therefore no stock solution was prepared. Expsoure duration was 21 days.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In order to achieve a saturated solution in a continous operation as required for a flow through test, water was percolated through columns. By mean of a peristaltic pump, the testwater saturated with test substance (after a time of contact of about 1 hour) was continously fed into the exposure tanks containing the fishes. The glass columns 100 cm long and with a diameter of 14.5 cm were packed as follows (in duplicate):

- layer 1 (10 cm): testwater
- layer 2 (10 cm): Polyethylene Terephtalate (PET) pearls
- layer 3 (70 cm): 1 g test substance / kg PET pearls
- layer 4 (10 cm): PET pearls

Polyethylene terephtalate (PET) was chosen as a filler because the test substance showed a good adherence on it. From a pretest it could be shown that this material showed no effect on fish and did not release any material into the water in detectable amounts. At the flow rate the contact time was calculated to be 1 hour. The column for the blank was packed with 90 cm PET without test substance. The columns for the test concentrations were prepared in duplicate.

- Evidence of undissolved material: No, the saturated solution was colorless and clear

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: P. Hohler / CH-4314 Zeiningen

- Length at study initiation: 86.5 mm (74-98 mm, 30 fish)
- Weight at study initiation: 7.14 g (4.64-11.59 g, 30 fish)
- Feeding during test: Food : NAFAG No.: 730 (Nähr- und Fischfutter AG, CH-9202 Gossau, Switzerland); 4 % of initial body weight daily given in two feeds

ACCLIMATION
- Acclimation duration: 85 days
- Acclimation conditions: Food : NAFAG No.: 730 (Nähr- und Fischfutter AG, CH-9202 Gossau, Switzerland) approximately 1 % of body weight daily, approximately 1 % of body weight live tubifex worms once a week.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

176 - 204 mg/L CaCO3

Test temperature:

15 ± 2 °C

pH:

7.9 - 8.3

Dissolved oxygen:

91 - 102 % saturation

Nominal and measured concentrations:

Nominal: Saturated solution

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria of 20 litres filled with 15 litres. (36/22/25 cm), front covered with paper to minimise disturbance
- Aeration: gentle aeration in the tanks during exposure
- Type of flow-through: peristaltic
- Flow rate: 3.75 L/h

- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.79 g/L/day at test start; 1.07 g/L/day at test end

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated (carbon filter) aerated tap water renewed 6 times per day.
- Intervals of water quality measurement: Every 2 to 3 days

OTHER TEST CONDITIONS
- Photoperiod: 16h daily

EFFECT PARAMETERS MEASURED: Mortality, growth parameters, swimming behaviour, exophtalmus, pigmentation/colour, food intake, reaction to external stimuli

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Extremely low water solubility

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: sublethal effetcs

Remarks on result:

other: No effects occur within the range of solubility.

Duration:

21 d

Dose descriptor:

LOEC

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: sublethal effects

Remarks on result:

other: No effects occur within the range of solubility.

Details on results:

- Mortality: None
- Sublethal effects: None

Reported statistics and error estimates:

Mortality
NOEC : The COCHRAN-ARMITAGE trend test (MANTEL, 1963) was used to consider either an increasing or decreasing number of dead fish with increasing concentration. If this trend test is not statistically significant then the analysis stops and the highest concentration is the NOEC, otherwise the highest concentration is discarded from the data and the procedure is repeated until the test for trend is no longer significant. The highest remaining concentration of the data is the NOEC. This procedure preserves Type I error.
Because the fish are identified on an individual basis (freeze branding) at the start and at the end of the exposure, the growth, condition, and conversion indices can be calculated for fish on an individual basis. These data are then treated as independent and analysed using classical statistical methods. For the calculation of rates a test duration of 21 days was assumed for all fish.
NOEC : Preliminary investigation of the data indicated that the data were nonnormal with skewed and possibly heavy tailed distribution. Because tests based upon normal theory approximations may lose substantial power in this case, a nonparametric analysis was used to test for a trend with increasing concentration level. Namely, JONCKHEERE's test (LEHMANN, 1975) was used to test for either increasing or decreasing values with increasing concentration level.

Validity criteria fulfilled:

yes

Conclusions:

In a long-term (21 days), flow-through fish toxicity study, the substance did not cause any effects using a saturated solution.

Executive summary:

The long-term fish toxicity of the substance was investigated in a GLP-study similar to OECD TG 204 exposing rainbow trout for 21 days.

Due to the very low water solubility, a saturated solution was tested under flow-through conditions (without analytical measurements).

Per control and treatment, 10 fish were used per treatment and control (dechlorinated tap water).

Measuring/observing mortality, growth parameters, swimming behaviour, exophtalmus, pigmentation/colour, food intake and reaction to external stimuli, the test substance did not induce any toxic effect.

Reference 2

Endpoint:

adult fish: sub(lethal) effects

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 OECD Guideline 204 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:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

Version / remarks:

Version from 1984

Deviations:

yes

Remarks:

The test concentration was test water saturated with test substance; the saturation was achieved using a flow through system; therefore no stock solution was prepared. Expsoure duration was 21 days.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In order to achieve a saturated solution in a continous operation as required for a flow through test, water was percolated through columns. By mean of a peristaltic pump, the testwater saturated with test substance (after a time of contact of about 1 hour) was continously fed into the exposure tanks containing the fishes. The glass columns 100 cm long and with a diameter of 14.5 cm were packed as follows (in duplicate):

- layer 1 (10 cm): testwater
- layer 2 (10 cm): Polyethylene Terephtalate (PET) pearls
- layer 3 (70 cm): 1 g test substance / kg PET pearls
- layer 4 (10 cm): PET pearls

Polyethylene terephtalate (PET) was chosen as a filler because the test substance showed a good adherence on it. From a pretest it could be shown that this material showed no effect on fish and did not release any material into the water in detectable amounts. At the flow rate the contact time was calculated to be 1 hour. The column for the blank was packed with 90 cm PET without test substance. The columns for the test concentrations were prepared in duplicate.

- Evidence of undissolved material: No, the saturated solution was colorless and clear

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: P. Hohler / CH-4314 Zeiningen

- Length at study initiation: 86.5 mm (74-98 mm, 30 fish)
- Weight at study initiation: 7.14 g (4.64-11.59 g, 30 fish)
- Feeding during test: Food : NAFAG No.: 730 (Nähr- und Fischfutter AG, CH-9202 Gossau, Switzerland); 4 % of initial body weight daily given in two feeds

ACCLIMATION
- Acclimation duration: 85 days
- Acclimation conditions: Food : NAFAG No.: 730 (Nähr- und Fischfutter AG, CH-9202 Gossau, Switzerland) approximately 1 % of body weight daily, approximately 1 % of body weight live tubifex worms once a week.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

176 - 204 mg/L CaCO3

Test temperature:

15 ± 2 °C

pH:

7.9 - 8.3

Dissolved oxygen:

91 - 102 % saturation

Nominal and measured concentrations:

Nominal: Saturated solution

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria of 20 litres filled with 15 litres. (36/22/25 cm), front covered with paper to minimise disturbance
- Aeration: gentle aeration in the tanks during exposure
- Type of flow-through: peristaltic
- Flow rate: 3.75 L/h

- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.79 g/L/day at test start; 1.07 g/L/day at test end

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated (carbon filter) aerated tap water renewed 6 times per day.
- Intervals of water quality measurement: Every 2 to 3 days

OTHER TEST CONDITIONS
- Photoperiod: 16h daily

EFFECT PARAMETERS MEASURED: Mortality, growth parameters, swimming behaviour, exophtalmus, pigmentation/colour, food intake, reaction to external stimuli

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Extremely low water solubility

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: sublethal effetcs

Remarks on result:

other: No effects occur within the range of solubility.

Duration:

21 d

Dose descriptor:

LOEC

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: sublethal effects

Remarks on result:

other: No effects occur within the range of solubility.

Details on results:

- Mortality: None
- Sublethal effects: None

Reported statistics and error estimates:

Mortality
NOEC : The COCHRAN-ARMITAGE trend test (MANTEL, 1963) was used to consider either an increasing or decreasing number of dead fish with increasing concentration. If this trend test is not statistically significant then the analysis stops and the highest concentration is the NOEC, otherwise the highest concentration is discarded from the data and the procedure is repeated until the test for trend is no longer significant. The highest remaining concentration of the data is the NOEC. This procedure preserves Type I error.
Because the fish are identified on an individual basis (freeze branding) at the start and at the end of the exposure, the growth, condition, and conversion indices can be calculated for fish on an individual basis. These data are then treated as independent and analysed using classical statistical methods. For the calculation of rates a test duration of 21 days was assumed for all fish.
NOEC : Preliminary investigation of the data indicated that the data were nonnormal with skewed and possibly heavy tailed distribution. Because tests based upon normal theory approximations may lose substantial power in this case, a nonparametric analysis was used to test for a trend with increasing concentration level. Namely, JONCKHEERE's test (LEHMANN, 1975) was used to test for either increasing or decreasing values with increasing concentration level.

Validity criteria fulfilled:

yes

Conclusions:

In a long-term (21 days), flow-through fish toxicity study, the substance did not cause any effects using a saturated solution.

Executive summary:

The long-term fish toxicity of the substance was investigated in a GLP-study similar to OECD TG 204 exposing rainbow trout for 21 days.

Due to the very low water solubility, a saturated solution was tested under flow-through conditions (without analytical measurements).

Per control and treatment, 10 fish were used per treatment and control (dechlorinated tap water).

Measuring/observing mortality, growth parameters, swimming behaviour, exophtalmus, pigmentation/colour, food intake and reaction to external stimuli, the test substance did not induce any toxic effect.

Description of key information

In a long-term (21 days), flow-through fish toxicity study, the substance did not cause any effects using a saturated solution.

Additional information

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

Note also that a long-term toxicity study in fish would not have been required, if it had not existed already, as the hazard assessment revealed neither a need to classify the substance as dangerous to the environment, nor that it is a PBT or vPvB substance, nor that there are any further indications that the substance may be hazardous to the environment (see Annex I of Regulation (EC) No 1907/2006).