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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:
31 January - 24 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13th April 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
April 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000
Version / remarks:
Paris September 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Date of production: 22 February 2016
Expiry date: 22 February 2021
Analytical monitoring:
yes
Details on sampling:
A supersaturated solution (100 mg/L nominal loading) was prepared by adding an amount of 0.0703 g test item to 703 mL test medium (ISO Medium) in the first renewal period and 0.0705 g test item in 705 mL test medium (ISO Medium) in the second renewal period one day before the start of each renewal period. The test solution was handled by ultrasonic bath for 10 minutes, thereafter stirred for a period of 24 hours to achieve an equilibrated concentration. The test solution was then filtrated through a membrane filter (0.45 µm; Nalgene® membrane, see section 5.1.3) to separate the possible non-dissolved test material. The test solution was freshly prepared in the testing laboratory just before introduction of the test animals (start of each renewal period of the experiment). Untreated control group (ISO Medium without test item) was included in the test.
As the test item is proved not to be stable for 48 hours in ISO Medium, the test solutions were renewed on day 1.
Vehicle:
no
Remarks:
Reconstituted water (ISO Medium)
Details on test solutions:
Test Solution Preparation

As the test item is poorly soluble in deionized water as well in the test medium, for preparation of test solution the water-accommodated fraction (WAF) approach was taken, as described in OECD Guidance Document No. 23. A supersaturated solution (100 mg/L nominal loading) was prepared by adding an amount of 0.0703 g test item to 703 mL test medium (ISO Medium) in the first renewal period and 0.0705 g test item in 705 mL test medium (ISO Medium) in the second renewal period one day before the start of each renewal period. 
The test solution was handled by ultrasonic bath for 10 minutes, thereafter stirred for a period of 24 hours to achieve an equilibrated concentration. The test solution was then filtrated through a membrane filter (0.45 µm; Thermo Nalgene® membrane*) to separate the possible non-dissolved test material. The test solution was freshly prepared in the testing laboratory just before introduction of the test animals (start of each renewal period).


Untreated Control

The test medium (ISO Medium) without test item added was used as control solution.

Reference Control

For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 392-202-3265) with reference item Potassium dichromate was: 05 – 06 September 2017.
The 24h EC50 value was determined to be 1.53 mg/L (95% conf. limits: 1.30 – 1.78 mg/L), which falls within the range of 0.6 mg/L and 2.1 mg/L as given in the guideline.

Name and data of Reference Item
Name: Potassium dichromate (K2Cr2O7)
Batch No.: MKBN3524V
Description: Orange crystalline powder
Expiry Date: 04 March, 2018
Storage: Room temperature
Supplier: SIGMA-ALDRICH

Test Water (Dilution Water)

Reconstituted water (ISO Medium, according to OECD 202) was used as dilution water in the experiment. Separate stock solutions of individual substances were first prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 water purification system). The ISO Medium was prepared by adding 25 mL from each of four stock solutions to one litre deionised water.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Age of parental stock: Less than 24 hours old
- Feeding during test: no

ACCLIMATION
- Acclimation period: no
- Acclimation conditions: same as test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
Animals were exposed to the test concentration or included as control over a period of 48 hours. The test animals were not fed during the test. Immobility or mortality was observed by visually after 24 and 48 hours.
Post exposure observation period:
no
Hardness:
The measured hardness of the ISO Medium used in the study was 249.2 mg/L (as CaCO3) in the first renewal period and 231.4 mg/L (as CaCO3) in the second renewal period.
Test temperature:
The test temperature was in the range of 20.4 - 20.5°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 20.3 – 20.8°C.
pH:
The pH of the test solution was not adjusted and did not vary by more than 1.5 units in any one test. The pH was in the range of 7.88 - 8.24 during the test.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 6.94 - 8.47 mg/L during the test.
Salinity:
not applicable
Conductivity:
not applicable
Nominal and measured concentrations:
Nominal test item concentration was 100 mg product/L.
Measured concentration (geometic mean): 0.5 mg/L
Details on test conditions:
The test vessels were kept in a climate chamber under controlled conditions during the test and test solutions were not aerated. The water temperature, dissolved oxygen concentration and pH were measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of each renewal period. Additionally, the ambient temperature was measured continuously using a min/max thermometer in the climate chamber.
Test type: Semi-static test. Based on the results obtained during analytical method validation (Study number: 805-100-2578) the test item is not stable for the duration of 48 hours in ISO Medium and not remains within 80 – 120% in comparison to that of the measured test item concentration at the start of the test throughout exposure. Therefore the test solution was renewed once during the test (on day 1).

Temperature: The test temperature was in the range of 20.4 - 20.5°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 20.3 – 20.8°C.

Oxygen Concentration: The dissolved oxygen concentration was in the range of 6.94 - 8.47 mg/L during the test.

pH: The pH of the test solution was not adjusted and did not vary by more than 1.5 units in any one test. The pH was in the range of 7.88 - 8.24 during the test.
Light-Dark Cycle: The experiment was carried out in complete darkness, as described by OECD 202.

Hardness: The measured hardness of the ISO Medium used in the study was 249.2 mg/L(as CaCO3) in the first renewal period and 231.4 mg/L (as CaCO3) in the second renewal period.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested at least twice a year.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF).
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 0.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF).
Details on results:
Validity of the Study

Immobilisation was not observed in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was in the range of 6.94 - 8.47 mg/L (more than 3 mg/L in all cases). All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Analytical Results

The concentration of the test item was analytically determined based on total product at the start and at the end of each renewal period of the experiment.

The test item was not detected in the untreated control group (i.e. signal intensities measured for the control samples were less than 20 % of the analytical quantification limit (LOQ). In the treated group the mean measured test item concentration was 0.600 mg/L at the start and 0.438 mg/L at the end of the first renewal period (corresponding to 73 % of the initial measured test concentration) while it was measured to be 0.557 mg/L at the start and 0.435 mg/L at the end of the second renewal period (corresponding to 78 % of the initial measured test concentration). The exposure concentration was calculated as the geometric mean of the start and end values and determined to be 0.50 mg/L. This concentration was considered as the saturation concentration in the test medium, based on water-accommodated fractions of the test item (equivalent to 100 mg/L nominal concentration).

Biological Results and Performance of the Test

Testing units were kept in a climate chamber at a temperature of 20.3 – 20.8 °C, while temperature in the test glasses ranged between 20.4 - 20.5 °C.
The pH was measured as 7.88 - 8.24 and the dissolved oxygen concentration between 6.94 - 8.47 mg/L during the study. The test was carried out in total darkness.
There was no immobilisation observed in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.
Results with reference substance (positive control):
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 392-202-3265) with reference item Potassium dichromate was: 05 – 06 September 2017.
The 24h EC50 value was determined to be 1.53 mg/L (95% conf. limits: 1.30 – 1.78 mg/L), which falls within the range of 0.6 mg/L and 2.1 mg/L as given in the guideline.
Reported statistics and error estimates:
A limit test was performed and toxic effects were not observed, therefore statistical analysis was not necessary. The NOEC and LOEC values of the test item were determined directly from the raw data.

Summary of the Biological Endpoints

Endpoints

Concentration1

48 h-NOEC

0.50 mg/L

48 h-LOEC

0.50 mg/L

1Concentration based on the mean measured concentration of the water-accommodated fraction of test item.

pH-values Measured during the Experiment

Test Group

Replicate

1strenewal period

2ndrenewal period

0 h

24 h

24 h

48 h

Control

1

8.05

8.09

8.08

8.23

2

8.17

8.24

3

8.16

8.24

4

8.16

8.20

Saturated test
concentration
(0.50 mg/L measured)

1

7.88

8.06

8.11

8.12

2

8.04

8.12

3

8.06

8.09

4

8.05

8.13

 

 Dissolved Oxygen Concentrations Measured during the Experiment

Test Group

Replicate

1strenewal period

2ndrenewal period

0 h

24 h

24 h

48 h

Control

1

8.41

8.30

8.47

8.39

2

8.21

8.27

3

8.16

8.22

4

8.11

8.18

Saturated test
concentration
(0.50 mg/L measured)

1

6.94

8.10

7.30

8.17

2

8.25

8.20

3

8.27

8.21

4

8.23

8.23

Temperatures Measured during the Experiment

Test Group

Replicate

1strenewal period

2ndrenewal period

0 h

24 h

24 h

48 h

Control

1

20.5

20.5

20.4

20.5

2

20.5

20.5

3

20.4

20.4

4

20.5

20.5

Saturated test
concentration
(0.50 mg/L measured)

1

20.5

20.5

20.5

20.4

2

20.5

20.5

3

20.5

20.5

4

20.4

20.5

Immobilization of the Test Animals

Test Group

Replicate

Number of
treated animals

Number of immobilised animals

24 h

48 h

Control

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

Saturated test
concentration
(0.50 mg/L measured)

1

5

0

0

2

5

0

0

3

5

0

0

4

5

0

0

 

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of this study, the test item Leuco Sulfur Blue 15 had no toxic effect up to aquatic saturation (i.e. limit test concentration) on Daphnia magna; the 48h LOEC is higher than the solubility level of the test item in the test medium, which corresponds to a mean measured concentration of 0.50 mg/L (based on total product). The NOEC value is equal to the mean measured test item concentration of 0.50 mg/L (based on total product). The EC50 was determined to be > 0.5 mg/L.
Executive summary:

Acute toxicity of the test item Leuco Sulfur Blue 15 was assessed on Daphnia magna in a 48-hour Acute Immobilisation Test. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours in a semi-static test. A limit test was performed in which, the test animals were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).

The quantification of the test item Leuco Sulfur Blue 15 was performed by a previously validated analytical method by the analytical laboratory of TOXI-COOP ZRT. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment and analysed by UV/VIS spectrophotometric method. The mean determined exposure concentration was 0.50 mg/L (based on total product) which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods.

Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the control. Each test vessel contained approximately 80 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium.

The immobilisation of the test animals was recorded 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of each renewal period. All measured values remained within the acceptable ranges.

There was no immobilisation in twenty daphnids exposed to either test item treated or control group. The EC50 was determined to be > 0.5 mg/L. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. All validity criteria were met and therefore the study can be considered as valid.

Description of key information

In a Daphnia sp. Acute Immobilisation Test according to OECD Guideline 202 with a WAF, the test item Leuco Sulfur Blue 15 had no toxic effect up to aquatic saturation (i.e. limit test concentration); the 48h LOEC is higher than the solubility level of the test item in the test medium, which corresponds to a mean measured concentration of 0.50 mg/L (based on total product). The NOEC value is equal to the mean measured test item concentration of 0.50 mg/L (based on total product). The EC50 was determined to be > 0.5 mg/L.

Key value for chemical safety assessment

Additional information

Acute toxicity of the test item Leuco Sulfur Blue 15 was assessed on Daphnia magna in a 48-hour Acute Immobilisation Test. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours in a semi-static test. A limit test was performed in which the test organisms were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).

The quantification of the test item Leuco Sulfur Blue 15 was performed by a previously validated analytical method by the analytical laboratory of TOXI-COOP ZRT. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment and analysed by UV/VIS spectrophotometric method. The mean determined exposure concentration was 0.50 mg/L (based on total product) which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods.

Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the control. Each test vessel contained approximately 80 mL test medium. Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test medium.

The immobilisation of the test animals was recorded 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of each renewal period. All measured values remained within the acceptable ranges.

There was no immobilisation in twenty daphnids exposed to either test item treated or control group. The EC50 was determined to be > 0.5 mg/L. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. All validity criteria were met and therefore the study can be considered as valid.