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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:
24-05-2017 - 26-05-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed using standard test guidelines
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
no
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
The stock solution (50 mg/L) was prepared by dissolving yellow powder in reconstituted water. Solution was kept in ultrasonic bath for 20 min. The test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test 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): The animals used for the test shall be less than 24 h old and should not be first brood progeny
- 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:
no
Total exposure duration:
48 h
Remarks on exposure duration:
± 1 hr
Test temperature:
20 ± 1°C
pH:
Without adjustment
Sample at concentration 1 mg/L: pH= 8.0 changed to pH 7.9 during the test
Control pH= 8.0 did not change during the test
Dissolved oxygen:
higher than 7.7 mg/L at the end of test
Nominal and measured concentrations:
0, 0.06, 0.12, 0.25, 0.50 and 1 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): 4

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)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.42 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 0.32 - 0.55 mg/L
Results with reference substance (positive control):
- Results with reference substance valid
- EC50: 0.73 mg/L (24 hours)
Validity criteria fulfilled:
yes
Conclusions:
The median effective concentration (EC50) for the test substance,4-(phenylazo)benzene-1,3-diamine, in Daphnia magna was determined to be 0.42 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 4-(phenylazo)benzene-1,3-diamine according to OECD Guideline 202.

The test substance was tested at the concentrations 0, 0.06, 0.12, 0.25, 0.50 and 1 mg/L. Effects on immobilisation were observed for 48 hours.

The median effective concentration (EC50) for the test substance, 4-(phenylazo)benzene-1,3-diamine, in Daphnia magna was determined to be 0.42 mg/L for immobilisation effects.

This value indicates that the substance is likely to be hazardouss to aquatic invertebrates and can be classified as Aquatic acute category 1 as per the CLP criteria.

Description of key information

The median effective concentration (EC50) for the test substance,4-(phenylazo)benzene-1,3-diamine, in Daphnia magna was determined to be 0.42 mg/L on the basis of mobiity inhibition effects in a 48 hour study.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.42 mg/L

Additional information

Different studies, experimental and predicted, of the substance 4-(phenylazo)benzene-1,3-diamine were reviewed for Short term toxicity to aquatic invertebrate endpoint and are summarised as follows:

Determination of the inhibition of the mobility of daphnids was carried out with the substance4-(phenylazo)benzene-1,3-diamineaccording to OECD Guideline 202. The test substance was tested at the concentrations0, 0.06, 0.12, 0.25, 0.50 and 1 mg/L. Effects on immobilisation were observed for 48 hours. The median effective concentration (EC50) for the test substance,4-(phenylazo)benzene-1,3-diamine, in Daphnia magna was determined to be 0.42 mg/L for immobilisation effects.

Supporting above data, Range finding study to Daphnia magna was carried out for 24 hrs. in scientific journal (Aquatic Toxicology, 2011). Neonates (3rd brood) were taken for the experiment. The test neonates were of <24h old. The test chemical conc. used for the study were 0.0248, 0.1243, 0.248, 0.497 and 0.746 mg/l, respectively (0.1, 0.5, 1, 2 and 3 µM). The Chlorella vulgaris was used to feed the D. magna (derived from a stock cultured at the Culture Collection of Algae and Protozoa (CCAP) Argyll, Scotland)were maintained in Bold’s Basal Medium; 0.431 mM K2HPO4, 1.29 mM KH2PO4, 0.304 mM MgSO4·7H2O, 2.94 mM NaNO3, 0.17 mM CaCl2·2H2O, 0.428 mM NaCl, 0.428 mM EDTA-Na4, 1.38 mM KOH, 44.8µM FeSO4·7H2O, H2SO4, 0.462 mM H3BO3,4.9µM ZnSO4·7H2O, 1.17µM MnCl2·4H2O, 1.1µM CuSO4·5H2O,0.275µM Co(NO3)2·6H2O, 0.79µM Na2MoO4·2H2O in a fermentervessel at 20±1◦C, under a 16-h:8-h light : dark cycle with light providedby a Gro-Lux photosynthetic tube.To provide a food stock, cells were collected by centrifugation(2250×g, 30 min, 20◦C) and re-suspended in a volume of waterto give an OD (1:10) of 0.8 at 440 nm. Groups of 10 neonates (<24 h old) in 100 ml fresh media were exposed to increasing concentrations of test chemical for 24 h (n= 2 test groups per concentration). After 24 h, any mortality and abnormal behaviour or appearance was recorded. The test was performed under the following conditions- pH-6 - 8 pH, Hardness-140 – 250 mg/l as CaCO3,Temp.-20 ± 1°C ,Conductivity-360–480 µS cm−1.Viability was analyzed by the number alive and speed of movement as compared to the control cultures. Based on percentage viability of test organism Daphnia magna, the LC0 and LOEC value was found to be 0.497 and 0.746 mg/l, respectively.

Further supporting data was based on the QSAR prediction done using the EPI Suite ECOSAR version 1.11 in which the 48 hours EC50 was estimated to be 0.778 mg/L on Daphnia Magna for substance 4-(Phenylazo)benzene-1,3-diamine with immobilization effects.

Thus considering the key and supporting values, it can be concluded that the substance is likely to be hazardous to aquatic invertebrates and can be classified as Aquatic acute category 1 as per the CLP criteria. But considering the harmonised classification of the substance and for the purpose of classification and chemical safety assessment the substance4-(phenylazo)benzene-1,3 -diamine is considered to classified as Aquatic acute 1 and aquatic chronic 1 category.