Reaction mass of Disodium dihydroxy[2-{[5-(hydroxy-kO)-3-methyl-1-(4-sulfophenyl)-1H-pyrazol-4-yl]diazenyl-kN1}benzoato(3-)-kO]chromate(2-) and Sodium hydroxy[2-{[5-(hydroxy-kO)-3-methyl-1-(4-sulfophenyl)-1H-pyrazol-4-yl]diazenyl-kN1}benzoato(3-)-kO]chromate(1-)

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

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

Principles of method if other than guideline:

None

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TCC/02/2014/CR/002
- Expiration date of the lot/batch: 23 February 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4 °C, in the dark.

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A nominal amount of test item (200 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 18, 32 and 56 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia Magna
- Source: In-house laboratory cultures
- Feeding during test: The Daphnids recieved no food during the exposure.

QUARANTINE (wild caught)

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Not applicable

Post exposure observation period:

None

Hardness:

Theoretical hardness of 250 mg/L as CaCO3.

Test temperature:

22 °C

pH:

8.0 - 8.3

Dissolved oxygen:

7.8 - 8.7 mg O2/L

Salinity:

None

Conductivity:

None

Nominal and measured concentrations:

Nominal: 0 (negative control), 10, 18, 32, 56, 100 mg/L
Measured (0 hours): 0 (Measured (48 hours): 0 (

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Type: Covered to reduce evaporation
- Material, size, headspace, fill volume: 150 mL with a fill volume of 100 mL
- Aeration: No
- Type of flow-through: Not applicable
- Renewal rate of test solution: Not applicable
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: Not applicable
- Biomass loading rate: Not measured

TEST MEDIUM / WATER PARAMETERS
Reconstituted Water – ISO Medium
Ingredient Final Concentration
(mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 65
KCl 5.8

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark, with 20 minutes dawn and dusk transition periods.
- Light intensity: between 200 and 1200 Lux (light periods)

EFFECT PARAMETERS MEASURED: Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10, and 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

A positive control was run as a separate study between the 29th February and 02nd March 2016.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence limits

Remarks:

50 - 72 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not recorded
- Other biological observations: Not applicable
- Mortality of control: None
- Other adverse effects control: Not applicable
- Abnormal responses: Not applicable
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None

Results with reference substance (positive control):

A positive control (Envigo Study Number MS29MC) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using the ToxRat Professional computer software package based on the nominal test concentrations gave the following results:

Time Point EC50 95 % Confidence Limits No Observed Effect Concentration (NOEC) Lowest Observed Effect Concentration (LOEC)
(Hours) (mg/L) (mg/L) (mg/L) (mg/L)
24 0.83 0.70 - 0.98 0.56 1.0
48 0.64 Not possible to determine 0.56 1.0

The No Observed Effect Concentration is based upon equal to or less than 10 % immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Range-finding Test

No immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, immobilization was observed at 100 mg/L.

Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 102 to 103% of nominal concentrations. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Definitive Test

Verification of Test Concentrations

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 90% to 106% of nominal and so the results are based on nominal test concentrations.

Immobilization Data

Nominal Concentration (mg/L)	Observations (Initial Population: 10 Per Replicate)
	24 Hours		48 Hours
	Cumulative Immobilized Daphnia	Observations	Cumulative Immobilized Daphnia	Observations
Control	0	10 N	0	10 N
0.10	0	10 N	0	10 N
1.0	0	2 T 8 N	0	1 T 9 N
10	0	1 T 9 N	0	2 T 8 N
100	6	2 T 2 R	9	1 R

N = No sub-lethal effects observed

T = Trapped at surface

R = Reduced mobility

A/I = All daphnia immobilized

The No Observed Effect Concentrations after 24 and 48 hours exposure were 100 and 18 mg/L respectively. The Lowest Observed Effect Concentration after 48 hours exposure was 32 mg/L.

The slopes and their standard errors of the response curves at 24 and 48 hours were 0.28 (SE = 11) and 5.3 (SE = 0.037) respectively.

Sub-Lethal Effects

Sub-lethal effects of exposure were observed in the 18 mg/L test concentration and above. These responses were daphnids being covered in debris, trapping at the surface and reduced mobility.

Validation Criteria

The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Observations on Test Item Solubility

At the start of and throughout the test all control solutions were observed to be clear colorless solutions, all 10 mg/L solutions were observed to be a light yellow solution, all 18 mg/L solutions were observed to be a yellow solution. All of the 32 mg/L solutions were observed to be a dark yellow solution, all 56 mg/L solutions were observed to be a light orange solution and all of the 100 mg/L solutions was observed to be an orange solution.

Validity criteria fulfilled:

yes

Conclusions:

Exposure to freshwater invertebrate Daphnia magna to FAT 20041/D TE has been investigated and gave the following results:
48h EC50 = 60 mg/L (95 % Confidence Limits = 50-72 mg/L); NOEC (48h) = 18 mg/L; LOEC (48h) = 32 mg/L.

Executive summary:

Introduction

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.

Methods

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

Results…….

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 90 % to 106 % of nominal and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item gave the following results:

Time Point (Hours)	EC50 (mg/L)	95 % Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	60	50	-	72	18	32

Description of key information

The 48-Hour EC50 = 60 mg/L (95 % Confidence Limits = 50-72 mg/L); NOEC (48 hours) = 18 mg/L; LOEC (48 hours) = 32 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

60 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. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 22 °C under static test conditions. The number of immobilized daphnids were recorded after 24 and 48 hours. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 90 % to 106 % of nominal and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item gave the following results:

48-Hour EC₅₀ = 60 mg/L (95 % Confidence Limits = 50-72 mg/L); NOEC (48 hours) = 18 mg/L; LOEC (48 hours) = 32 mg/L.