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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts
EC Number:
812-037-7
Cas Number:
1793011-72-9
Molecular formula:
Unknown for all components
IUPAC Name:
Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts
Test material form:
solid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L (nominal)
- Sampling method: Samples were taken at 0 and 72 hours after exposure at each concentration and analysed by HPLC.

Test solutions

Vehicle:
no
Details on test solutions:
A stock solution of 100 mg/L was prepared by adding the appropriate amount of substance to 1 L of test media in a Mess flask. This was stirred for 5 minutes. This stock solution was then diluted as appropriate to provide the remaining test concentrations (50 mL test solutions in 500 mL erlenmeyer flasks with aluminium caps).

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella Subcapitata
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection
- Method of cultivation: Maintained by periodic subculture using Gorham medium. Algae are kept under sterile conditions.

ACCLIMATION
- Acclimation period: 4 days
- Any deformed or abnormal cells observed: No

Study design

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

Test conditions

Hardness:
Not specified
Test temperature:
21.3 to 22.1°C throughout the test
pH:
7.8 to 7.9 throughout the test
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L
Measured (mean): 0.961, 3.15, 9.92, 30.5 and 102 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flask with aluminium cap
- Type: open (although capped with aluminium)
- Material, size, headspace, fill volume: 50 mL
- Aeration: No
- Initial cells density: 5 x 10^3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium as detailed in the respective guideline)

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 110 to 120 µE/m2/s

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cells/mL (biomass) and growth rate

TEST CONCENTRATIONS
- Range finding study: First test (100 mL/vessel) Control, 1.0, 10 and 100 mg/L. Second test (50 mL/vessel) Control, 1, 5, 10 and 100 mg/L.
- Test concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Results used to determine the conditions for the definitive study: As the substance is black and water soluble the range-finder was conducted at an increased light intensity with test volumes of 50 or 100 mL. The results showed high toxicity at 100 mg/L in both experiments which decreased through the concentrations. It was determined that the growth inhibition of algae was indeed affected by the attenuation of light as a result of the test substance colour. The inhibitory effect was less pronounced when using test solution volumes of 50 mL and accordingly this was used in the main test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
29.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: effect likely caused by the attenuation of light from the coloured substance rather than a true toxic effect
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: effect likely caused by the attenuation of light from the coloured substance rather than a true toxic effect
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: Control vessels were colourless, 1 and 3.2 mg/L were indigo and 10, 32 and 100 mg/L were black.
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
Growth inhibition tests with a reference reagent potassium dichromate are conducted every 6 months. The ErC50 values obtained in the most recent test were within historical ranges for tests conducted at the laboratory.
Reported statistics and error estimates:
The EC50 was determined using the least squares linear regression analysis of points recognised as straight in the concentration-inhibition curves. 95% confidence limits were calculated where possible. The NOEC was determined by an analysis of variance (ANOVA), Williams test, subsequent to Bartlett test for homogeniety of variance.

Any other information on results incl. tables

Biomass in the control cultures increased exponentially above 16 fold during the 72 hour culture. All validity criteria with regards to the controls were met.

The ability of the substance to affect light transmission was seperately investigated. This showed that light transmission was greatly reduced where the test substance was used as a filter causing high increases in growth inhibition. This showed that the toxicity observed in the definitive phase was likely to be as a result of the substance's ability to block light transmission to algal cells.

Growth inhibitions (%) of Pseudokirchneriella subcapitata

Test group

Nominal [Mean measured] mg/L

Vessel No.

Growth rate

Rate µ (0-72 hr)

Inhibition (%)

Iµ ( 0-72 hr)

Control

NA

1

0.0720

NA

2

0.0694

3

0.0739

4

0.0722

5

0.0692

6

0.0712

Average

0.0713

NA

SD

0.0018

Conc. 1

1.0 [0.61]

1

0.0754

-5.8

2

0.0747

-4.8

3

0.0717

-0.6

Average

0.0739

-3.7

SD

0.0020

Conc. 2

3.2 [3.15]

1

0.0658

7.7

2

0.0720

-1.0

3

0.0692

2.9

Average

0.0690

3.2

SD

0.0031

Conc. 3

10 [9.92]

1

0.0565

20.8

2

0.0604

15.3

3

0.0549

23

Average

0.0573**

19.7

SD

0.0028

Conc. 4

32 [30.5]

1

0.0342

52

2

0.0349

51.1

3

0.0360

49.5

Average

0.0350

50.9

SD

0.0009

Conc. 5

100 [102]

1

0.0104

85.4

2

0.0096

86.5

3

0.0116

83.7

Average

0.0105**

85.2

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Validity criteria on test fulfilled
Conclusions:
The 72 hour ErC50 of the test substance was determined to be 29.3 mg/L with the corresponding NOEC as 3.15 mg/L. However, the effects observed were most likely a result of the substance's properties (black coloured) which attenuated light minimising the potential for photosynthesis of the algae.