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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-28 to 2018-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Remarks:
All concentrations levels and the control were analytically verified via HPLC-DAD
Details on sampling:
- Concentrations: 100, 50.0, 25.0, 12.5 and 6.25 % of a saturated solution were analytically verified
- Sampling method: The samples and the control were diluted with acetonitrile containing 0.2% H3PO4 directly after sampling (dilution factor 1.43). The lower concentrated samples (6.25% to 25.0% of the saturated solution) and the control were analyzed without further dilution. The other concentrations (50.0% and 100% of the saturated solution) were diluted with dilution medium. Additionally, samples from the end of the exposure were analyzed without dilution.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading of 100 mg/L test item was prepared with dilution water 24 hours prior to the start of the exposure. The test item was weighed out and the solution was stirred 24 hours at room temperature, 1100 rpm. Undissolved particles were removed by membrane filtration (membrane filter 0.45 pm, RC, Macherey-Nagel). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate was used in the test. During filtration, the filter was always kept covered. The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. The solution was clear. Therefore, it can be concluded that the test item was completely dissolved. The saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: :6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution.
- Controls: Negative control: Six replicates (without test item) were exposed under the same conditions as the test concentrations. Positive control: Potassium dichromate was tested as a reference item.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: HINDAK, SAG 61.81
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z- Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
Nominal range: 21 - 24 “C, controlled at ± 2 °C
pH:
7.5 ± 1
Nominal and measured concentrations:
Nominal: 100, 50.0, 25.0, 12.5, 6.25 % of a saturated solution
Geometric measured mean: 1.12, 0.792, 0.553, 0.389, 0.273 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks
- Material, size, headspace, fill volume: Sterile Erlenmeyer flasks (vol. 250 mL), fill volume 100 mL
- Aeration: not specified
- Initial cells density: 6839 celIs/mL
- Control end cells density: 1541423 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per 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
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 Iux, corresponding to 60 to 120 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell growth and yield at 0, 24, 48 and 72 h
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the preliminary range finding test at the saturated solution.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Test concentrations: Nominal 100, 10 and 1 mg/L saturated solution
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.09 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.722 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.553 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.493 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.767 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.389 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: growth: 0.559 CI (0.538-0.581); yield: 0.320 CI (0.242-0.341)
Reported statistics and error estimates:
EC10-, EC20- and EC50-value with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal 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 p-value (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05.

Any other information on results incl. tables

Table 1: Nominal and measured concentrations for 72 h

% saturated solution

m-Xylylenebismaleimide

 

Measured concentration [mg/L] 0 h

Measured concentration [mg/L] 72 h

Geometric mean measured concentration [mg/L]1)

100

16.8

< LOQ

1.12

50

8.37

< LOQ

0.792

25

4.08

< LOQ

0.553

12.5

2.02

< LOQ

0.389

6.25

0.991

< LOQ

0.273

control

< LOQ

< LOQ

 

1)      = Measured concentrations < LOQ were taken into account with half of the LOQ

Table 2: Effect of m-Xylyenebismaleimide on the cell density and growth rate of Pseudokirchneriella subcapitata

Treatment  concentration (mg/l)

Initial cell density

Cell density at
72h

Treatment
Mean cell density

% Inhibition of cell density

Growth rate
(0h to 72h)

Treatment
Mean growth rate

% Inhibition of growth rate

Nominal

Measured*

Rep

Dilution control

1

 6839

1437120

1541423 

 

1.78

 

 

2

 6839

1671786

 

 

1.83

 

 

3

 6839

1562997

 

 

1.81

 

 

4

 6839

1377781

 

 

1.77

 

 

5

 6839

1675648

 

 

1.83

 

 

6

 6839

1523203

 

 

1.80

 

 

6.25

0.273

1

 6839

1514894

1540974 

 

1.80

2

 6839

1545441

 

 

1.81

 

 0

3

 6839

1562587

 

 

1.81

 

 0

12.5

0.389

1

 6839

1394401

 1547450

 

1.77

 0

 2

2

6839 

1702166

 

 

1.84

 

 -2

3

 6839

1645792

 

 

1.61

 

 0

25.0

0.553

1

 6839

1145223

 1264165

 

1.71

 4

 5

2

 6839

1294917

 

 

1.75

 

 3

3

 6839

1352325

 

 

1.76

 

 2

50.0

0.792

1

 6839

598997

717344 

 

1.49

 14

 17

2

 6839

922613

 

 

1.64

 

 9

3

 6839

630422

 

 

1.51

 

 16

100.0

1.12

1

6839

71310

76925

 

0.78

55

57

 

 

2

6839

72943

 

 

0.79

 

56

 

 

3

6839

8693

 

 

0.85

 

53

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study,m-Xylylenebismaleimide was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured concentrations): The EC50-value with 95% confidence intervals after 72 h was 1.09 mg/L (1.04 - 1.11). The EC10-value with 95% confidence intervals after 72 h was 0.722 mg/L (0.663 - 0.764). The NOEC-value after 72 hours was 0.553 mg/L.