1,1'-(4-methyl-1,3-phenylene)bis-1H-pyrrole-2,5-dione

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-24 to 2017-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-DAD

Details on sampling:

- Concentrations: The concentrations of the test item were determined at the start of the exposure (0 hours) of all tested concentrations and the control and after 24 hours of the highest test item concentration.
- Sample storage conditions before analysis: The samples and the control were stabilized factor 2 with acetonitrile containing 0.2% H3PO4 directly after sampling. Further dilutions were made prior to analysis with acetonitrile : HPLC-water (30 : 70) containing 0.1% H3PO4.

Vehicle:

yes

Remarks:

DMF (Dimethylformamide)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the preparation of the test concentrations of the test item, appropriate stock solutions were prepared in DMF (solvent). With regard to the properties of the test item (i.e. the fast hydrolysis rate) an aprotic solvent has been chosen to prepare the test concentrations, The stock solutions were prepared in measuring flasks (10 mL capacity) by direct weighing of appropriate amounts of the test item into the measuring glass flasks.
Thereafter, the flasks were filled up with DMF to 10 mL. The stock solutions were treated with ultrasound for 1 minute at room temperature. The test concentrations were prepared by adding 0.100 mL/L of the appropriate stock solutions to the dilution water. These solutions were shaken manually and used for testing.
- Controls: Negative control (without test item), Solvent control, positive control: Potassium dichromate is tested twice a year as reference item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMF (Dimethylformamide)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.100 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No precipitation observed.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Desmodesmus subspicatus CHODAT SAG 86.81
- Age of inoculum (at test initiation): Fresh stocks were prepared every month on Z-Agar.
- Method of cultivation: Cultured on Z-Agar. Light intensity amounted to 2567 -- 5130 lux for 24 h per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test medium

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Nominal range: 21 - 24°C, controlled at ± 2°C

pH:

7.38 to 7.48 at study initiation and 7.78 to 8.18 at study end

Nominal and measured concentrations:

Nominal concentrations: 0.625, 1.25, 2.50, 5.00, 10.0 mg/L
Initial measured concentrations: 0.667, 1.33, 2.64, 5.28, 10.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
- Type (delete if not applicable): closed
- Initial cells density: Nominal: approximately 2 - 5 E03 cells/mL; Actual: 5836 cells/mL
- Initial cells density: 5836 cells/mL
- Control end cells density: 1957434 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Threefold concentrated AAP medium according to the guidelines
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH of the test medium had to be 7.5 ± 0.1 and was adjusted prior to testing with addition of 1 M NaOH and HCl.
- Photoperiod: 24 hours/day light
- Light intensity and quality: 4440 to 8680 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer; colorimeter
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 665 nm. Dilution water was used as background signal.
- Other: Microscopic evaluation of the cells at the start and end of the exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometrical series with a dilution factor of 2
- Range finding study
- Test concentrations: 0.0200, 0.200, 2.00, 10.0 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.64 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

5.28 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 10.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 10.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.667 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.667 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.667 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

< 0.667 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
growth: 0.697, CI: 0.647-0.755, Valid Range: 0.668 ± 0.375
yield: 0.325, CI: 0.308-0.343, Valid Range: 0.318 ± 0.130

Reported statistics and error estimates:

EC10, EC20 and EC50-value with confidence intervals of yield inhibition after 72 hours was calculated by slgmoidal dose-response regression. The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’S test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α=0.05.

The measured concentrations of the test item at the start of the exposure were in the range of 106 to 107% of the nominal values. After 24 hours of the exposure, the highest concentration of the test item was < LOQ of the nominal values. Therefore it can be considered that the concentrations of the test item at the end of the exposure were below LOQ, too.

Table 1: Nominal and measured concentrations of the test item:

Sampling date	Start of the exposure 0 h		24 h
Nominal test item concentration [mg/L]	2,4-Bismaleimidotoluene
	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]
10.0	10.6	106	< LOQ
5.00	5.28	106	n.d.
2.50	2.64	106	n.d.
1.25	1.33	106	n.d.
0.625	0.667	107	n.d.
Control	< LOQ		n.d.
Solvent control	< LOQ		n.d.

Table 2: Evaluation after 72h:

Initially measured test item concentration [mg/L]	Replicate No.	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield[%]
10.6	Mean	1.68	7	906679	46
5.28	Mean	1.74	4	1077259	36
2.64	Mean	1.81	0	1308132	22
1.33	Mean	1.72	5	1011234	40
0.667	Mean	1.80	1	1288456	23
Solvent Control (0.100 ml- DMF/L)	Mean	1.81		1671719
Control	Mean	1.86		1950080

Table 3: Cell Densities:

Initially measured test item concentration[mg/L]	ReplicateNo.	0 hours	Cell density [cells/mL]   24 hours	48 hours	72 hours
10.6	Mean	5836	22887	136196	912515
5.28	Mean	5836	30339	173720	1083095
2.64	Mean	5836	33939	205138	1313968
1.33	Mean	5836	36631	162145	1017070
0.667	Mean	5836	37626	204756	1294292
Solvent Control (0.100 ml- DMF/L)	Mean	7354	52026	232087	1679073
Control	Mean	7354	43757	249930	1957434

Validity criteria fulfilled:

yes

Conclusions:

In this study 2,4-Bismaleimidotoluene was found to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 10.6 mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were > 10.6 mg/L and < 0.667 mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 2.64 and < 0.667 mg/L, respectively.
All effect levels are given based on the initially measured test item concentration of 2,4-Bismaleimidotoluene.

Description of key information

- OECD guideline study (201, adopted 2011), fresh water green alga Desmodesmus subspicatus was exposed to nominal concentrations of 0.625, 1.25, 2.50, 5.00, 10.0 mg/L corresponding to initial measured concentrations of 0.667, 1.33, 2.64, 5.28, 10.6 mg/L for 72h under static conditions. EC50 = > 10.6 mg/L, EC10 = >10.6 mg/L and NOEC (growth) = 2.64 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

10.6 mg/L

EC10 or NOEC for freshwater algae:

10.6 mg/L

Additional information

In a study conducted according to OECD TG 201 freshwater green alga Desmodesmus subspicatus was exposed to 2,4 -Bismaleimidotoluene at nominal concentrations of 0.625, 1.25, 2.50, 5.00, 10.0 mg/L corresponding to initial measured concentrations of 0.667, 1.33, 2.64, 5.28, 10.6 mg/L for 72h under static conditions.

2,4-Bismaleimidotoluene was found to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 10.6 mg/L, respectively. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were > 10.6 mg/L and < 0.667 mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 2.64 and < 0.667 mg/L, respectively.

All effect levels are given based on the initially measured test item concentration of 2,4-Bismaleimidotoluene.