6-amino-4-hydroxynaphthalene-2-sulphonic acid

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6-amino-4-hydroxynaphthalene-2-sulphonic acid

EC Number:

202-000-8

EC Name:

6-amino-4-hydroxynaphthalene-2-sulphonic acid

Cas Number:

90-51-7

Molecular formula:

C10H9NO4S

IUPAC Name:

6-amino-4-hydroxynaphthalene-2-sulphonic acid

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominally)
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.

Test solutions

Vehicle:

no

Remarks:

medium was used

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared to give the desired series of test concentrations. 200.8 mg of the test item were added to 2 litres of dilution water and treated for 1 h in an ultrasonic bath. The pH was measured to be 6.3. To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.562 mL of the algal inoculum were added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton stoppers.
- Controls: medium without any test item
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): see attachment 2

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).

ACCLIMATION
- Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Culturing media and conditions (same as test or not): Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium (attachment 2).
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium (attachment 2) to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Hardness:

Water hardness of the final nutrient medium was 1.3 °dH, corresponding to 22.5 mg/L CaCO3.

Test temperature:

21 - 24 °C

pH:

7.0 - 8.1 (start of the study); 7.5 - 8.7 (end of the study)

Nominal and measured concentrations:

Nominal: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Measured: 0.369 mg/L, 1.043 mg/L, 3.332 mg/L, 9.906 mg/L, 34.532 mg/L and 105.91 mg/L after 0 h; 0.099 mg/L, 0.351 mg/L, 0.763 mg/L, 1.716 mg/L, 5.012 mg/L and 40.768 mg/L (42.660 mg/L without algae) after 72 h

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with cotton stoppers, test volume: 100 mL
- Initial cells density: approximately 5000 cells per millilitre
- No. of vessels per concentration (replicates): 3; additionally highest test concentration without algae. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, at all measuring points (0, 24, 48 and 72 hours) a test flask without algae was run in parallel to the geometric series of test concentrations.
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (OECD medium of OECD TG 201 was used for the growth of the algae in the pre cultures and the preparation of stock and test solutions of the test item; see attachment 2)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium (attachment 2).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.
- Determination of cell concentrations: Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced

TEST CONCENTRATIONS
- Range finding study: A range finding test preceded the main test and provided information about the range of concentrations which were used in the main test.
- Test concentrations: 0.01, 0.1, 1.0 and 10 mg/L

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.49 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to EU method C.3, which is equivalent to OECD guideline 201, on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards algae.
The toxicity against planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) was tested in a static test at nominal test concentrations of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Results based on growth rate after 72h:
EC50 = 10.73 mg/L (7.0 -18 mg/L 95% confidence limits)                 
EC10 = 0.45 mg/L (0.14 -0.91 mg/L 95% confidence limits)
NOEC = 0.59 mg/L                      
LOEC = 1.49 mg/L
Based on these results, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, taking into account the fact that the test item is not readily biodegradable, the test item should be classified as aquatic chronic Cat. 3.

Executive summary:

A GLP-study was performed to assess the adverse effects of Gammasäure on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to nominally 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of Gammasäure dissolved in dilution water. Auxiliary used to prepare the test media was an ultrasonic bath.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to Williams Multiple Sequential t-test Procedure. The following values were determined [mg/L]:

ErC 50* (0-72 h): 10.73

ErC 10* (0-72 h): 0.45

NOEC [r]* (tα 0.05): 0.59

LOEC [r]* (tα 0.05): 1.49

After 24 hours of exposure, the test media coloured to brown in the test item concentrations 3.686 mg/L, 12.991 mg/L and 63.987 mg/L (nominal 10-100 mg/L). No brown colour was visible over the test period at the test item concentrations of 0.196 mg/L, 0.589 mg/L and 1.492 mg/L (nominal 0.32-1.0 mg/L).

NOEC/ErC 10 were determined at test concentrations which showed no coloration, while the ErC 50 was determined at concentrations which showed coloration and for which an influence on the result may not be excluded.

The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 99.1 % to 115 % of nominal values at 0 hours, from 41.3 to 81.1 % of nominal values at 24 hours, from 26.3 % to 62.1 % of nominal values at 48 hours and from 15.7 % to 42.7 % of nominal values at 72 hours.