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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:
07 January 2019 to 22 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Daphnia fed on algal suspension and GM 300 suspension at the range finding test and not algal suspension and a Tetramin suspension. GM 300 has been previously tested to confirm that it is a suitable alternative food source to Tetramin for Daphnia.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
Daphnia fed on algal suspension and GM 300 suspension at the range finding test and not algal suspension and a Tetramin suspension. GM 300 has been previously tested to confirm that it is a suitable alternative food source to Tetramin for Daphnia.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%
Analytical monitoring:
yes
Remarks:
HPLC with UV-VIS detection
Details on sampling:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass vessels containing 100 mL Elendt M7 medium (see Annex 2) 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 an algal suspension (Desmodesmus subspicatus) and GM300 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.

A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test. Details of the most recent positive control (Envigo study number KC41PP) are given in Annex 3.

Test Water
Reconstituted water (Elendt M7 medium) used for the range-finding test, initial experiment and definitive test is defined in Annex 2.

Experimental Design and Study Conduct

3.3.1 Preliminary Media Preparation Trial
Preliminary solubility work indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019). Therefore, a media preparation trial was conducted in order to determine the solubility of the test item under test conditions (see Annex 4).

3.3.2 Range-finding Test
The results obtained from the preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.11 mg/L could be obtained using a saturated solution method of preparation.
The test concentration to be used in the initial experiment was determined by a preliminary range-finding test. In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes, to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the numbers of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were analyzed immediately. However, analysis of these samples was unsuccessful as low results were obtained for the procedural recovery samples. As no duplicate samples were taken during the range finding test, a separate stability trial was performed alongside the validation of the method to assess test item stability in the absence of the test organism.

3.3.3 Initial Experiment
The range-finding test indicated that there was no toxicity at the highest concentration of 100% v/v saturated solution, therefore an initial experiment was conducted as a limit test at a test concentration of 100% v/v saturated solution.
The results of the initial experiment showed that at the 100% v/v saturation test group all of the daphnids were immobilized, therefore a further experiment utilizing a full range of test concentrations was required.
The results from the initial experiment were not used for reporting purposes.

3.3.4 Definitive Test
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes, to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 6.25, 12.5, 25 and 50% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 48 hours (see Annex 5).

3.3.4.1 Exposure Conditions
As in the range-finding test 150 mL glass vessels containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed in each test and control vessel at random, containing the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period.

Vehicle:
no
Details on test solutions:
Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.
A preliminary media preparation trial indicated that a dissolved test item concentration of 0.11 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of aqueous solubility of the test item in Elendt M7 media under test conditions.
Following a preliminary range-finding test and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution for 48 hours at a temperature of 20 °C to 21 °C under static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by centrifugation at 40000 g for 30 minutes, to give a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

1.3 Results
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.025 to 0.31 mg/L. Analysis of the test preparations at 48 hours showed that the measured test concentrations had declined, ranging from less than the limit of quantification (LOQ = 0.011 mg/L) to 0.11 mg/L. Therefore, it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.012, 0.013, 0.042, 0.082 and 0.19 mg/L.

Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
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.
Test temperature:
20 °C to 21 °C
pH:
7.9 to 8.6
Dissolved oxygen:
8.8 to 9.1
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25, 50, 100
Measured concentrations (geometric mean): 0.012, 0.013, 0.042, 0.082, 0.19
Details on test conditions:
Water Quality Criteria
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

3.4.3 Verification of Test Concentrations
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 taken for immediate analysis. Duplicate sets of samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in Annex 5.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 0.012 - <= 0.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.012 - <= 0.19 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
There was no significant immobilization in 20 daphnids exposed to geometric mean measured test concentrations of 0.012, 0.013, 0.042, 0.082, and 0.19 mg/L for a period of 48 hours.
Inspection of the immobilization data gave the following results based on the geometric mean measured concentrations:

Time (hour) EC50 (mg/L) 95% Confidence limits (mg/L)
24 >0.19 Not determined
48 >0.19 Not determined

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.19 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

6.2.3 Sub-Lethal Effects
No sub-lethal effects of exposure were observed throughout the definitive test
Results with reference substance (positive control):
Analysis of the immobilization data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

Time point (Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC (mg/L)
24 1.1 0.95 - 1.3 0.56 1.0
48 0.80 0.12 - 1.5 0.56 1.0

A level of 20% immobilization was recorded in the 0.32 mg/L test concentration; however, as the immobilization recorded in the 0.56 mg/L test concentration was only 10% immobilization it was considered unlikely that the effect observed at 0.32 mg/L was as a result of exposure to the reference item and therefore the NOEC was 0.56 mg/L.
The NOEC 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*.

*The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24-Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L
Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 0.19 mg/L. The No Observed Effect Concentration (NOEC) was 0.19 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, in accordance with GLP.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.025 to 0.31 mg/L. Analysis of the test preparations at 48 hours showed that the measured test concentrations had declined, ranging from less than the limit of quantification (LOQ = 0.011 mg/L) to 0.11 mg/L. Therefore, it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.012, 0.013, 0.042, 0.082 and 0.19 mg/L.

Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 0.19 mg/L. The No Observed Effect Concentration (NOEC) was 0.19 mg/L.

Description of key 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, in accordance with GLP.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.025 to 0.31 mg/L. Analysis of the test preparations at 48 hours showed that the measured test concentrations had declined, ranging from less than the limit of quantification (LOQ = 0.011 mg/L) to 0.11 mg/L. Therefore, it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.012, 0.013, 0.042, 0.082 and 0.19 mg/L.

Exposure of Daphnia magna to the test item gave EC50 values based on the geometric mean measured test concentrations of greater than 0.19 mg/L. The No Observed Effect Concentration (NOEC) was 0.19 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.19 mg/L

Additional information