Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 June 2017 - 27 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 3 November 2015

Specific details on test material used for the study:

Stability in water: stable

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the limit concentration (100 mg/L):
Frequency: at t=0 h and at t=48 h
Volume: 2.0 mL from the approx. centre of the test vessels
Storage: Samples were stored in a freezer (≤ 15°C) until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct application to the test medium: the test item was not completely soluble in test medium at the loading rates initially prepared. Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A one day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were filtered through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations.
- Controls: Test medium without test item or other additives.
- Evidence of undissolved material: solutions were checked for Tyndall effect with a laser pen. No distraction of the beam was observed. All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Age at study initiation: <24 hours
- Source: in-house laboratory culture with a known history, at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions. The daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age of parental stock: >2 weeks
- Feeding during test: no

BREEDING
- Breeding method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. Cultures (maximum age of 4 weeks) were renewed after 7 days of cultivation by renewing the medium twice a week.
- Feeding: daily, a suspension of fresh water algae.
- Health during breeding period: no mortality observed

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

180 mg CaCO3/L

Test temperature:

20-21°C

pH:

Start: 7.7 (control) and 8.0 (test item)
End: 8.1

Dissolved oxygen:

Start: not determined
End: 9.0 mg/L

Nominal and measured concentrations:

Nominal concentration: WAF prepared at a concentration of 100 mg/L (limit concentration)
Measured concentration: 0.184 mg/L at t=0; 0.178 mg/L at t=48

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass vessels, open, fill volume: 80 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 for the highest test concentration, 2 for the other test concentrations
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by Reverse Osmosis
- Culture medium different from test medium: yes, test medium is adjusted ISO medium
- Intervals of water quality measurement: at t=48 h (dissolved oxygen and pH measurements). The dissolved oxygen concentrations and pH were not measured at the start of the test. However, pH was measured in the highest WAF at the end of the preparation procedure and in ISO medium used as the control treatment, just before test initiation and was within range described in the study plan. Oxygen was > 3.0 mg/L at the end of the exposure period. It was therefore assumed that it was at similar levels at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours daily

EFFECT PARAMETERS MEASURED: immobility at t=24 and t=48 hours

COMBINED LIMIT/RANGE-FINDING STUDY
- Test concentrations: WAFs prepared at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: since no effects on mobility were observed, the results from the 100 mg/L test solution in the combined limit/range-finding test were used as the final study.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (June 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.18 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- No effects on mobility of the daphnids were seen at the limit concentration tested.
- Since the measured concentration at the end of the exposure period was 97% of the initial measured concentration at t=0, the effect parameter was based on the intitial measure concentration.
- After 48 hours of exposure to the test item, 10% and 5% immobility was observed in WAFs prepared at 10 mg/L and 100 mg/L, respectively. However, these effects were biologically irrelevant (≤10%).
- No abnormalities were observed during the exposure period.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Concentrations tested: 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
- Relevant effect levels: 48h-EC50: 0.59 mg/L, 95% CI: 0.52-0.66 mg/L
- Other: the results where within the expected range according to the historical data (>0.3 and <1.0 mg/L)

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks' section.

Conclusions:

The 48h-EC50 for Daphnia magna exposed to D&C Red 6 was >0.18 mg/L based on the initial measured exposure concentration.

Executive summary:

In a short-term toxicity test, performed according to OECD guideline 202 and GLP principles, D&C Red 6 was assessed for its potential toxicity towards Daphnia magna. Twenty daphnids were exposed to a blank control and a limit concentration (WAF prepared at 100 mg/L) each, whereas 10 daphnids were exposed to 1.0 and 10 mg/L each in a combined limit/range-finding test under static conditions for 48 hours.

Since the test item was poorly soluble, WAFs were individually prepared and the filtered test solutions were used in the test. Samples were taken from the control and the highest concentration at the start and at the end of the test for analysis. Results showed that the concentration at t=0 h was 0.18 mg/L and that this concentration remained stable during the exposure period (97% of the initial measured concentration at t=48 h). Therefore, the effect concentration was based on the initial measured concentration.

Incidence of immobility was recorded at t=24 and t=48. Since no biologically relevant effect was observed during the exposure period at any of the concentrations, the 48h-EC50 for Daphnia magna exposed to D&C Red 6 was determined to be >0.18 mg/L.

The acceptability criteria were met and the study was considered to be valid.

Description of key information

In a short-term toxicity test, performed according to OECD guideline 202 and GLP principles, D&C Red 6 was assessed for its potential toxicity towards Daphnia magna.Twenty daphnids were exposed to a blank control and a limit concentration (WAF prepared at 100 mg/L) each, whereas 10 daphnids were exposed to 1.0 and 10 mg/L each in a combined limit/range-finding test under static conditions for 48 hours.

Since the test item was poorly soluble, WAFs were individually prepared and the filtered test solutions were used in the test. Samples were taken from the control and the highest concentration at the start and at the end of the test for analysis. Results showed that the concentration at t=0 h was 0.18 mg/L and that this concentration remained stable during the exposure period (97% of the initial measured concentration at t=48 h). Therefore, the effect concentration was based on the initial measured concentration.

Incidence of immobility was recorded at t=24 and t=48. Since no biologically relevant effect was observed during the exposure period at any of the concentrations, the 48h-EC50 for Daphnia magna exposed to D&C Red 6 was determined to be >0.18 mg/L.

The acceptability criteria were met and the study was considered to be valid.