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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19.10.2016-21.10.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experiment test result performed using standard test guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Principles of method if other than guideline:
Short term toxicity to aquatic invertebrates were performed according to the OECD guideline 202 in a static system.
GLP compliance:
no
Specific details on test material used for the study:
Name of test material (as cited in study report): Amaranth dye
Molecular formula (if other than submission substance): C20H11N2Na3O10S3
Molecular weight (if other than submission substance): 604.47
Smiles notation (if other than submission substance): c1ccc2c(c1)c(ccc2S(=O)(=O)[O-])N=Nc3c4ccc(cc4cc(c3O)S(=O)(=O)[O-])S(=O)(=O)[O-].[Na+].[Na+].[Na+]
InChl (if other than submission substance): 1S/C20H14N2O10S3.3Na/c23-20-18(35(30,31)32)10-11-9-12(33(24,25)26)5-6-13(11)19(20)22-21-16-7-8-17(34(27,28)29)15-4-2-1-3-14(15)16;;;/h1-10,23H,
(H,24,25,26)(H,27,28,29)(H,30,31,32);;;/q;3*+1/p-3
Substance type: Organic
Physical state: Solid

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: No data
- Expiration date of the lot/batch: No data
- Purity test date: No data

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: No data
- Specific activity: No data
- Locations of the label: No data
- Expiration date of radiochemical substance: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data
Analytical monitoring:
not specified
Details on sampling:
No data available
Vehicle:
yes
Details on test solutions:
The solution 100 mg/l was prepared by dissolving brown powder in reconstituted water.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Source: Own breeding at University of Chemistry and Technology, Prague
- Age at study initiation (mean and range, SD):
- Feeding during test: No feeding

ACCLIMATION - No data available
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
± 1 hr
Post exposure observation period:
No data available
Hardness:
No data available
Test temperature:
20±1°C
pH:
Test: 7.8 (changed to 7.6 during test)
Control: 7.7 (change to 7.5 during test)
Dissolved oxygen:
higher than 7.9 mg/L at the end of test
Salinity:
No data available
Conductivity:
No data available
Nominal and measured concentrations:
Nominal 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 ml glass vessel
- fill volume: 25 ml
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 5

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Natural water (surface or ground water), reconstituted water or dechlorinated tap water are acceptable as culturing and dilution water if D. magna survives in it for the duration of the culturing, acclimation and testing without showing signs of stress. Waters in the range pH 6 to pH 9, with hardness between 140 mg/l and 275 mg/l (as CaCO3) are recommended.
As an example, the preparation of dilution water meeting the requirements is described below.
Dissolve known quantities of reagents in water. The dilution water prepared shall have a pH of 7.8 ± 0.5, a hardness of (225 ± 50) mg/l (expressed as CaCO3), a molar Ca + Mg ratio close to 4 + 1 and a dissolved oxygen concentration above 7 mg/l.

Prepare the solutions specified below:
- Calcium chloride solution: Dissolve 117.6 g of calcium chloride dihydrate (CaCl2.2H2O) in water (4.2) and make up to 1 l with water (4.2).
- Magnesium sulfate solution: Dissolve 49.3 g of magnesium sulfate heptahydrate (MgSO4.7H2O) in water (4.2) and make up to 1 l with water (4.2).
- Sodium bicarbonate solution: Dissolve 25.9 g of sodium bicarbonate (NaHCO3) in water (4.2) and make up to 1 l with water (4.2).
- Potassium chloride solution: Dissolve 2.3 g of potassium chloride (KCI) in water (4.2) and make up to 1 l with water (4.2).

Mixing
Mix 2.5 ml of each of the four solutions and make up to 1 l with water.
The dilution water shall be aerated until the dissolved oxygen concentration has reached saturation and the pH has stabilized. If necessary, adjust the pH to 7.8 ± 0.5 by adding sodium hydroxide (NaOH) solution or hydrochloric acid (HCI). The dilution water prepared in this way shall not be further aerated before use.

- Sodium hydroxide solution, e.g. [NaOH] : 1 mol/l.
- Hydrochloric acid, e.g. [HCl] : 1 mol/l.

Reference substance:
Dissolve 600 mg of potassium dichromate (K2Cr2O7) in water and make up to 1 l with water (4.2).

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustment done
- Photoperiod: No - Darkness
- Light intensity:

CALCULATION:
EC50 was calculated using non linear regression by the software Prism 4.0
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Duration:
48 h
Dose descriptor:
other: EC8
Effect conc.:
100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Nontoxic
Details on results:
100 mg/l of test chemical was used at which 8% daphnia inhibited after the exposure of 48 hrs.
Results with reference substance (positive control):
- Results with reference substance valid
- EC50: 0.79 mg/L (24 hours)
Reported statistics and error estimates:
No data
Validity criteria fulfilled:
not specified
Conclusions:
The effective concentration (EC8) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4-[(4-sulfo-1-naphthalenyl), sodium salt; Amaranth dye, in Daphnia magna was determined to be 100 mg/L on the basis of mobiity inhibition effects in a 48 hour study.
Executive summary:

Determination of the inhibition of the mobility of daphnids was carried out with the substance 2,7-Naphthalenedisulfonic acid, 3-hydroxy- 4-[(4-sulfo-1-naphthalenyl), sodium salt; Amaranth dye according to OECD Guideline 202. The limit test wasperformed at 100 mg/l. Effects on immobilisation were observed for 48 hours. The effective concentration (EC8) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4- [(4-sulfo-1 -naphthalenyl), sodium salt (Amaranth dye), in Daphnia magna was determined to be 100 mg/L on the basis of mobiity inhibition effects in a 48 hour study. This value indicates that the substance is likely to be non-hazardous to aquatic invertebrates and can not be classified as toxic as per the CLP criteria.

Description of key information

Determination of the inhibition of the mobility of daphnids was carried out with the substance 2,7-Naphthalenedisulfonic acid, 3-hydroxy- 4-[(4-sulfo-1-naphthalenyl), sodium salt; Amaranth dye according to OECD Guideline 202. The limit test wasperformed at 100 mg/l. Effects on immobilisation were observed for 48 hours. The effective concentration (EC8) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4- [(4-sulfo-1 -naphthalenyl), sodium salt (Amaranth dye), in Daphnia magna was determined to be 100 mg/L on the basis of mobiity inhibition effects in a 48 hour study. This value indicates that the substance is likely to be non-hazardous to aquatic invertebrates and can not be classified as toxic as per the CLP criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

From the available experimental data for the target chemical, the information is summarized as below:

In the first experimental data for the chemical chemical from ABITEC report, determination of the inhibition of the mobility of daphnids was carried out with the substance 2,7-Naphthalenedisulfonic acid, 3-hydroxy- 4-[(4-sulfo-1-naphthalenyl), sodium salt; Amaranth dye according to OECD Guideline 202. The limit test wasperformed at 100 mg/l. Effects on immobilisation were observed for 48 hours. The effective concentration (EC8) for the test substance, 2,7-Naphthalenedisulfonic acid, 3-hydroxy-4- [(4-sulfo-1 -naphthalenyl), sodium salt (Amaranth dye), in Daphnia magna was determined to be 100 mg/L on the basis of mobiity inhibition effects in a 48 hour study. This value indicates that the substance is likely to be non-hazardous to aquatic invertebrates and can not be classified as toxic as per the CLP criteria.

 

Similarly the toxic effects of Amaranth were studied (a screening method for the toxicity of food dyes using artemia salina larvae, 1977) on Artemia salina larvae. Artemia salina (A. salina eggs) a crustacean, commonly known as brine shrimp eggs, are commercially available, and are easily cultured in the laboratory because they are resistant to environmental stresses. Active larvae can be obtained within 1 to 2 days and no live culture is required for a few days thereafter. A salina eggs (encysted dried gastrulae) were commercially obtained, and were stored at -200°C. Eggs used in experiments were washed and stored at room temperature in a desiccators over anhydrous granular CaCl2. Larvae were obtained by incubating eggs in petri dishes containing muslin-filtered sea water at 30°C for 24 hours. The larvae were separated from shells, dead larvae and unhatched eggs by their phototactic movements towards a light source. Amaranth at concentrations of 6044.7mg/l and 604.47 mg/l were placed in a petri dish, and sea water containing 20 to 30 larvae was added. After this was incubated at 30°C for 24 hours and 48 hours, larvae surviving were measured by direct count. The same method was tested from 5 to 6 times for each concentration, and the death rate was calculated. Death was assumed to have occurred when there was no movement. The death rate was defined as the average of the percentage of deaths observed for 24 hours and 48 hours. 100% death rate was noted after 48 hours when 6044.7 mg/l of Amaranth was exposed to the test organism and 0% death rate after 24 hours in case of exposure to 604.47 mg/l of test chemical.

 

Structure-toxicity relationships (The Hydractinia echinata Test-System. III: Structure-Toxicity Relationship Study of Some Azo-, Azo-Anilide, and Diazonium Salt Derivatives, 2014) for Acid Red 27 were developed usingHydractinia echinata(H. echinata) as model species.Test chemical was purchased from leading chemical suppliers from their catalogues. Colonies of H. echinata (Biologische Anstalt, Helgoland, Germany) were used to obtain eggs and larvae. The culture medium was artificial seawater (980 mosmol, pH 8.2, 18 °C). In laboratory an artificial metamorphosis can be synchronically started by the introduction of Cs+ ions or by using seawater without Mg2+ ions; it then lasts only 24 h. Under the action of external stimulus of Cs+ ions or Cs+ ions together with the tested compounds, one part of larvae further lives as such and another one is metamorphosized to the polyp form. The evaluation of the influence of the tested substance is very clear this way, the proposed method being based on this aspect.H. echinatalarvae were exposed to seawater containing Cs+ and simultaneously one of the test substances for 3 h. The percentage of animals that underwent metamorphosis (development into polyps) was determined after 24 h. During the following days the frequency of inductions did not further increase. A concentration of inducers was chosen which caused about three half to three quarters of the larvae to metamorphose in order to have conditions which are highly sensitive against an inhibitory influence. The concentration of the test substances (expressed in mol/L) was varied in such a way that we were able to determine the concentration at which the frequency of induction was reduced by 50% with respect to a control. This concentration was termed MRC50 (forMetamorphosisReductionConcentration) and is similar to the effective EC50 concentration that gives half maximal effective response.The logarithm of the reciprocal value (log1/MRC50) values, the average (C) concentrations of these values was calculated for the test chemical. The MRC50value calculations result from the graphical representation of the metamorphosis variation, M (%), (Y axis) function of the xenobiotic’s concentration (mol/L) (X axis), where the metamorphosis decreases with the rise of the xenobiotic’s concentration. Thus, the MRC50value represents the xenobiotic’s concentration (mol/L) necessary for a 50% decrease of metamorphosis, with respect to control.The MRC 50 value for Hydractinia echinata after 3 hours of exposure to test chemical was determined to be 2827.360 mg/l. The test chemical can be considered non- toxic to Hydractinia echinata under the test conditions.

 

On the basis of available information for the target substance, the test substance cannot be considered hazardous to the aquatic organisms at environmentally relevant concentrations and cannot be classified as per the CLP classification.