2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, trisodium salt

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:

April 2018

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:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, An-nex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibi-tion Test”

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Regulation (EU) No. 2016/266 amending Regulation (EC) No. 440/2008, Annex IV, Method C.3:
“FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBI-TION TEST,” adopted 07. December 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

A stock solution containing 100.0 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. Lower test concentrations were prepared by dilution of the stock solution in algal medium

For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.469 mL) to achieve a cell concentration of 2.6E3 cells/mL. For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal preculture (0.820 mL).
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
In these mixtures, the pH-value was measured. Samples for the analytical determination were taken.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The concentration of the test item in the test vessels was measured at the start and at the end of the test (after determination of cell numbers).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

1 Specification
Unicellular freshwater green alga.
Genus Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
2 Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the preculture was prepared

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Four days before the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially

Hardness:

Resulting hardness in mmol/L: 2.502
Resulting hardness in mg CaCO3/L: 250

Test temperature:

21.2 – 22.1 °C

pH:

7.8 / 7.7

Conductivity:

< or = to 0.4 µS/cm

Nominal and measured concentrations:

Nominal measured

3.2 mg/L 2.63 mg/L
10 mg/L 9.24 mg/L
32 mg/L 31.90 mg/L
100 mg/L 98.39 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers covered with perforated plastic wrap
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 65 mL

- No. of organisms per vessel: 2600 cells/ml
- 6 replicates for the blank control
3 replicates for each treatment

GROWTH MEDIUM
- Standard medium used: yes



OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod:
- Light intensity and quality: 5000 lux
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

>= 4.55 - <= 7.35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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: no
- Flocculation: no
- Adherence to test vessels: 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: no

Nominal Concentra-tion in mg/L Parameter Cell Number/mL
0 h 24 h 48 h 72 h
Blank control Mean 2600 7907 27753 104223
Blank control SD 0 255 1051 3862
1 Mean 2600 7147 26273 105600
1 SD 0 914 2000 6505
3.2 Mean 2600 6907 27860 107907
3.2 SD 0 283 695 8619
10 Mean 2600 6680 22520 76893
10 SD 0 871 3490 6227
32 Mean 2600 5873 15793 29300
32 SD 0 484 1641 1178
100 Mean 2600 6013 17740 20800
100 SD 0 70 537 783

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.90 mg/L
Biological Results of K2Cr2O7
Parameter Value 95% confidence interval
72h ErC50 0.90 mg/L 0.77 – 1.1 mg/L
72h EyC50 0.42 mg/L 0.35 - 0.51 mg/L

Reported statistics and error estimates:

The measured concentrations lay between 82 % and 109% of the nominal concentra-tions. The lowest treatment lay below the LOQ. The other measured concentrations were all ± 20% of the nominal value. Nevertheless, the nominal values are used for statistical evaluation, since no inhibition has occurred in the two lowest treatments. As the QC-samples’ concentration deviated more than 3% and less than 10% from the nominal val-ue, the recovery rate of 95.7 % at the beginning and 92.3 % at the end of the test was in-cluded in the determination of the concentrations.
The measured blank values were below the LOQ, which is why they could not be deter-mined.

Microscopical Observations

Nominal Concentrationin mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
1	Yes
3.2	Yes
10	Yes
32	Yes
100	No cells visible

Validity criteria fulfilled:

yes

Conclusions:

a toxic effect of the test substance on the algal cells can be excluded up to a concentration of 100 mg/L.

Executive summary:

The study was performed according to the

The study was performed using 5 concentrations ranging from 1.0 to 100 mg/L. Incubation time (test systemDesmodesmus subspicatus)was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield[1] were determined from the cell number at the respective observation times.Significant inhibition of algal growth was observed at the three highest concentrations