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Toxicity to aquatic algae and cyanobacteria

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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:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.