Trisodium [μ-[3-[[4'-[[6-benzamido-1-hydroxy-3-sulpho-2-naphthyl]azo]-3,3'-dihydroxy[1,1'-biphenyl]-4-yl]azo]-4,5-dihydroxynaphthalene-2,7-disulphonato(7-)]]dicuprate(3-)

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:

2020

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 11127/E TE
Batch: 2024 (Thailand)
Purity: 63.1% total inorganics, 36.9% total organics
Physical State/Appearance: Blue solid
Expiry Date: 06 February 2024
Storage Conditions: Room temperature in the dark.

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and the 100 mg/L loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates (R1 to R4) at 48 hours for immediate quantitative analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

yes

Remarks:

water

Details on test solutions:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2019 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain/clone: Magna
- Age at study initiation: 24h old
- Source: Covance in-house laboratory
- Feeding during test : Each culture was fed daily with a mixture of algal suspension (Raphidocellis subcapitata) and GEMMA Micro 300 fish food suspension.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

19 to 20 degC

pH:

8.2 - 8.4

Dissolved oxygen:

8.6 - 9.0 mg O2/L

Nominal and measured concentrations:

Range-finding test: 1.0, 10 and 100 mg/L.
Definitive Test: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel
- Material, size, headspace, fill volume: 240 mL
- Volume of solution: 100mL
- Aeration: No
- No. of organisms per vessel: 5 daphnids per vessel
- No. of vessels per concentration: Four
- No. of vessels per control: 5 daphnids per vessel
- No. of vessels per vehicle control: Four

TEST MEDIUM / WATER PARAMETERS
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: If necessary the pH the pH was adjusted with NaOH or HCl.
- Photoperiod: 16 hours of light and 8 hours of dark.
- Light intensity: between 200 and 1200 lux

EFFECT PARAMETERS MEASURED : Immobilization.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test a "limit test" was conducted at a loading rate of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines no immobilization or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities:
- Mortality of control: none
- Immobilisation of control: 10% (2/20 daphnia were found to be immobilized).

Results with reference substance (positive control):

48h EC50 = 0.71 mg/L.

Reported statistics and error estimates:

An estimate of the EL50 values was given by inspection of the immobilization data

Range-finding Test

Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test. No immobilization or sub-lethal effects of exposure were observed throughout the test. Based on this information a single loading rate of four replicates of 100 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no

immobilization or adverse reactions to exposure were observed.

Chemical analysis of the test preparations at 0 hours (see Annex 4) showed measured test copper concentrations to range from 0.018 to 1.7 mg/L. There was no significant change in the measured concentrations at 48 hours.

Definitive test : Chemical Analysis of Test Loading Rates

Chemical analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured copper concentration of less than the limit of quantification (LOQ) of the analytical method employed was obtained which was determined to be 0.027 mg/L. Given that this was lower than expected based on previous analyses of a 100 mg/L loading rate WAF, the duplicate sample which had been stored frozen was analyzed which gave a measured copper concentration of 2.2 mg/L (equivalent to 74 mg/L as test item based on a test item copper content of 2.99%). Chemical analysis of the aged test preparation at 48 hours showed a measured copper concentration of 2.0 mg/L was obtained (equivalent to 68 mg/L as test item).

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on the nominal loading rate only.

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAF. At both the start and end of the mixing period, and following a 1-Hour standing period, the 100 mg/L loading rate WAF was observed to have formed an opaque dark purple media column. Visual examination of the WAF at the end of standing showed there to be dispersed test item present and therefore it was considered justifiable to remove the WAF by filtering through two glass wool plugs (2 to 4 cm in length) and two sheets of coarse filter paper to remove as much undissolved test item as possible. Visual observations after filtration showed that dispersed test item remained, however, it was considered that further filtration at this point would not have removed any more of the undissolved test item present. During the test the control preparations were observed to be clear colorless solutions whilst the 100 mg/L loading rate WAF preparations were observed to be very dark purple dispersions

Validity criteria fulfilled:

yes

Conclusions:

The EC50 of test item was found to be greater than 100 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 19 °C to 20 °C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

The concentration of copper in the test preparations was determined at 0 and 48 hours by copper analysis with inductively coupled plasma with mass spectrometric detection (ICP-MS) using an external standard.

Chemical analysis of the 100 mg/L loading rate WAF at 0 hours showed a measured copper concentration of 2.2 mg/L was obtained (equivalent to 74 mg/L as test item based on a test item copper content of 2.99%).

Chemical analysis of the aged test preparation at 48 hours showed a measured copper concentration of 2.0 mg/L was obtained (equivalent to 68 mg/L as test item).

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on the nominal loading rate only.

Exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading (NOEL) rate was 100 mg/L loading rate WAF.

Description of key information

Exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading (NOEL) rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, 20 daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 19 to 20 °C under static test conditions. No immobilization or adverse reactions to exposure were recorded after 24 and 48 hours.

Thus,exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading (NOEL) rate was 100 mg/L loading rate WAF.