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

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

Test material information

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 concentrations

Effect concentrations 1

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

Effect concentrations 2

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.