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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:
05 March 2018 to 07 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No.:202 (Adopted: 13 April 2004) OECD: Paris.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACh), Annex IV Part C, C.2 (published in the Official Journal of the European Union L 220 of 24 August 2009).
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
EPA Health Effects Test Guidelines, OCSPP 850.1010.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
no
Details on sampling:
As, no chemical analysis of the prepared formulations were performed as part of this study. Traceability (equipment used, quantities of Test Item weighed, etc.) of form preparations were checked and revealed no abnormalities of consequences. Furthermore, for this study, the formulations were prepared just before the treatment. Consequently, the absence of formulation analysis data was considered not to prejudice the overall GLP status of the study and the scientific reliability of the study conclusions.
Vehicle:
no
Details on test solutions:
Because the Test Item was very poorly soluble in water (See more details in study with Citoxlab study code: 17/080-345AN and 17/080-316AN), a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. A saturated Test Item solution at 100 mg/L Test Item nominal loading rate was prepared by dispersing/dissolving the needed amount of Test Item into the test medium (ISO media) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30°C and then was equilibrated for about 24 hours at approximately 20°C. The non-dissolved test materials were removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solution. As only Limit test was carried out, further dilution of this saturated solution was not performed.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species and strain: Daphnia magna
Source: Szent István University, Department of Aquaculture 2100 Gödöllő, Páter Károly u. 1. – Hungary
Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: There were 20 animals in test and control groups respectively, divided into 4 replicates (5 animals / replicate)
Age of the animals: They were less than 24 h old at the beginning of the test
Acclimatization: There was no acclimatization because the water used was similar to the culture water
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
The reconstituted water (ISO medium) had a total hardness of 249 mg/L (as CaCO3).
Test temperature:
The water temperature was measured at the start of the test and 24-hour intervals thereafter in each test vessel. The test temperature was in the range of 20.3 – 20.8°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 20.1 and 20.9°C.
pH:
The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of each renewal period in each test vessel and was in the range of 7.10 – 7.87.
Dissolved oxygen:
The dissolved oxygen concentration was measured in each test vessel at the start and at the end of each renewal period and was in the range of 7.2– 8.6 mg/L.
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Because significantly toxic response was not observed during the preliminary concentration Range-Finding Test, only one test concentration at 100% saturated solution and one control group was tested in a Limit Test.
Details on test conditions:
PERFORMANCE OF THE TEST
The test duration was 48 hours. Twenty animals, divided into four groups (glass beaker) of five animals each (at least 5 mL test solution/animal) were used at the test concentrations and for the control in a semi-static system. The animals were not fed during the test.
The choice of the test concentration was done on the basis of the results of a preliminary range-finding test.

Preliminary Range-Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the Test Item so that appropriate test concentrations could be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions.

OBSERVATIONS
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.
The water temperature, the oxygen concentrations and pH of the controls and the test solution were measured at the beginning and at the end of the renewal periods.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test item loading rate
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test item loading rate
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test item loading rate
Basis for effect:
mobility
Details on results:
VALIDITY
There was no immobilised animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
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: 17/367-023DA) with reference item Potassium dichromate (batch no.: A0345704) is: 06-07 December 2017. The 24h EC50: 0.65 mg/L, (95 % confidence limits: 0.61 – 0.70 mg/L).
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects. The EC50, NOEC, LOEC and EC100 values were determined directly from the raw data.

Result of the Preliminary Range-Finding Test

Nominal concentration

[mg/L test item loading rate]

Untreated control

0.1

1

10

100

Number of treated / immobilised animals

10 / 0

10 / 0

10 / 0

10 / 0

10 / 1

 

Number and percentage of immobilised animals

Test Group

Number of treated animals

Number of immobilised animals

24 hours

48 hours

Number

Percent

Number

Percent

Control

20

0

0

0

0

100 mg/L test item nominal loading rate

20

0

0

0

0

 

Temperature measured in the test vessels (°C)

Test group

Replicate

Measuring

0 h

(fresh media)

24 h

(old media)

24 h

(fresh media)

48 h

(old media)

Control

1

20.8

20.3

20.6

20.4

2

20.8

20.3

20.6

20.4

3

20.8

20.3

20.6

20.4

4

20.8

20.3

20.6

20.4

100 mg/L test item nominal loading rate

1

20.8

20.3

20.6

20.4

2

20.8

20.3

20.6

20.4

3

20.8

20.3

20.6

20.4

4

20.8

20.3

20.6

20.4

 

Oxygen concentration measured in the test vessels (mg/L)

Test group

Replicate

Measuring

0 h

(fresh media)

24 h

(old media)

24 h

(fresh media)

48 h

(old media)

Control

1

8.6

8.3

8.5

8.3

2

8.6

8.4

8.5

8.3

3

8.6

8.4

8.5

8.3

4

8.6

8.4

8.5

8.3

100 mg/L test item nominal loading rate

1

7.2

8.3

7.3

8.3

2

7.2

8.4

7.3

8.4

3

7.2

8.4

7.3

8.3

4

7.2

8.4

7.3

8.3

 

pH measured in the test vessels

Test group

Replicate

Measuring

0 h

(fresh media)

24 h

(old media)

24 h

(fresh media)

48 h

(old media)

Control

1

7.87

7.42

7.41

7.10

2

7.87

7.43

7.41

7.12

3

7.87

7.43

7.41

7.15

4

7.87

7.43

7.41

7.17

100 mg/L test item nominal loading rate

1

7.86

7.43

7.46

7.19

2

7.86

7.43

7.46

7.21

3

7.86

7.43

7.46

7.23

4

7.86

7.43

7.46

7.25

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the Daphnia magna acute immobilisation study observed endpoints for the effect of Test Item were the followings:
The 24h, 48h, EC50 value: > 100 mg/L test item nominal loading rate
The 48h EC100 value: > 100 mg/L test item nomninal loading rate
The 48h NOEC: 100 mg/L test item nominal loading rate
The 48h LOEC: > 100 mg/L test item nominal loading rate
Based on the results of this study, the Test Item had no toxic effect at saturation on the Daphnia; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium.
Executive summary:

Acute toxicity of Test Item was assessed with Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

 

Because no toxic effect was observed at the solubility level of the Test Item in the preliminary test, only only one test concentration at 100% saturated solution and one Control group were tested in a Limit test in the definitive study.

 

Since the Test Item was very poorly soluble in water (solubility level in water was <LOQ in water results obtained under the following Citoxlab Hungary study code: 17/080-345AN) and due to that there was no toxic effect at the solubility level of the Test Item in the test medium during the Preliminary Range-Finding Test the experiment was carried out without analytical measurements in agreement with the Sponsor.

 

Twenty animals, divided into 4 groups (glass beaker) of 5 animals each were used at the test and for the control groups. All validity criteria were met during this study.

 

Under the conditions of this Daphnia magna acute immobilisation study observed endpoints for the effect of Test Item were the followings:

 

The 24h, 48h, EC50 value: > 100 mg/L test item nominal loading rate

The 48h EC100 value: > 100 mg/L test item nominal loading rate

The 48h NOEC: 100 mg/L test item nominal loading rate

The 48h LOEC: > 100 mg/L test item nominal loading rate

 

Based on the results of this study, the Test Item had no toxic effect at saturation on the Daphnia; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium.

Description of key information

The 24h, 48h, EC50 value: > 100 mg/L test item nominal loading rate

The 48h EC100 value: > 100 mg/L test item nominal loading rate

The 48h NOEC: 100 mg/L test item nominal loading rate

The 48h LOEC: > 100 mg/L test item nominal loading rate

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

Acute toxicity of Test Item was assessed with Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

Because no toxic effect was observed at the solubility level of the Test Item in the preliminary test, only only one test concentration at 100% saturated solution and one Control group were tested in a Limit test in the definitive study.

Since the Test Item was very poorly soluble in water and due to that there was no toxic effect at the solubility level of the Test Item in the test medium during the Preliminary Range-Finding Test the experiment was carried out without analytical measurements in agreement with the Sponsor.

Twenty animals, divided into 4 groups (glass beaker) of 5 animals each were used at the test and for the control groups.

 

Under the conditions of the Daphnia magna acute immobilisation study observed endpoints for the effect of Test Item were the followings:

The 24h, 48h, EC50 value: > 100 mg/L test item nominal loading rate

The 48h EC100 value: > 100 mg/L test item nominal loading rate

The 48h NOEC: 100 mg/L test item nominal loading rate

The 48h LOEC: > 100 mg/L test item nominal loading rate

Based on the results of this study, the Test Item had no toxic effect at saturation on the Daphnia; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium.