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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 12 June 2017. Experimental completion date 04 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Identification: 1-(methylamino)-4-[(3-methylphenyl)amino] anthraquinone
Physical state/Appearance: dark red powder
Batch: 702W01
Purity: 99.4%
Expiry Date: 08 February 2020
Storage Conditions: room temperature in the dark
Intended use/Application: industrial chemical
Specific details on test material used for the study:
Identification: 1-(methylamino)-4-[(3-methylphenyl)amino] anthraquinone
Physical state/Appearance: Dark red powder
Batch: 702W01
Purity: 99.4%
Expiry Date: 08 February 2020
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range-finding Test
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 stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Samples were taken from the control and 100% v/v saturated solution test group from the freshly prepared bulk test preparation at 0 hours and from the old or expired pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Preparation of Test Samples
The test samples were thawed with the aid of a waterbath. A volume (5 mL) of test sample with the addition of water (5 mL) were pipetted into a volumetric flask (20 mL) and made to volume with acetonitrile.

Test solutions

Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial
Preliminary solubility work conducted 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 Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
Saturated Solution Preparation
A nominal amount of test item (550 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)
Discussion
It is evident from these results that a decline in measured concentrations occurred when the stirring period was extended beyond 24 hours. Whilst the centrifuged samples yielded the highest dissolved test item concentration, visual inspection of the 48-Hour sample centrifuged at 40000 g showed undissolved test item to be present. Therefore, it was considered that centrifugation was not sufficient to ensure all undissolved test item was removed.
Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 50 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded) to give a 100% v/v saturated solution of the test item.

Range-finding Test
A nominal amount of test item (550 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 filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) 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.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (550 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 filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL 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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Test temperature:
Temperature was maintained at 22 °C throughout the test
pH:
7.8 - 8.0
There were no treatment related differences for pH.
Dissolved oxygen:
8.2 - 8.8 mgO2/L
There were no treatment related differences for oxygen concentration.
Nominal and measured concentrations:
Range-finding Test
nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
Definitive Test
nominal test concentrations of 100% v/v saturated solution.

Measured test concentrations of less than the limit of quantification (LOQ)
Details on test conditions:
Range-finding Test
In the range-finding test 5 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 with 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 vessels were employed per concentration. 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 number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test
As in the range-finding test 150 mL glass beakers 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, in 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 with 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.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 other: 100% v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: 100% v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution. Some trapping was observed in the 0.10, 1.0 and 10% v/v saturated solution test concentrations at 48 hours, however, given that no effects were observed in the 100% v/v saturated solution test preparation, this was considered not to have had an impact on the outcome of the test.
Chemical analysis of the 100% v/v saturated solution test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.11 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.
Based on this information, a single test concentration of four replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at highest attainable test concentration, no immobilization or adverse reactions to exposure were observed.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.11 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.
There was no immobilization in 20 daphnids exposed to a test concentration of 100% v/v saturated solution for a period of 48 hours.
Exposure of Daphnia magna to the test item gave EC50 values based on the nominal test concentrations of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.
Sub-Lethal Effects
No sub-lethal effects of exposure were observed throughout the test.
Results with reference substance (positive control):
A positive control 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, however, throughout the definitive test the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 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:

At 24 hour time point
EC50 mg/L = 1.3
95% Confidence Limits (mg/L) = 1.0 - 1.8
No Observed Effect Concentration (NOEC) (mg/L) = 1.0
Lowest Observed Effect Concentration (LOEC) (mg/L) = 1.0

At 48 hour time point
EC50 mg/L = 1.2
95% Confidence Limits (mg/L) = 1.1 - 1.3
No Observed Effect Concentration (NOEC) (mg/L) = 0.56
Lowest Observed Effect Concentration (LOEC) (mg/L) = 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.

Any other information on results incl. tables

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.

Water Quality Criteria

Temperature was maintained at 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Throughout the test the light intensity was observed to be in the range 462 to 495 Lux.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the nominal test concentrations gave a 48-Hour EC50 value of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.
This study showed that there were no toxic effects at saturation.
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

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 saturated solution method of preparation was appropriate for this test item.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a nominal concentration of 100% v/v saturated solution for 48 hours at a temperature of 22 °C under static test conditions. The test item solutions were prepared by stirring an excess (50 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 filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.11 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Exposure of Daphnia magna to the test item gave EC50 values based on the nominal test concentrations of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution.

This study showed that there were no toxic effects at saturation.