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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:
15 January 2018 to 17 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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 (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to 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.3.
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:
yes
Details on sampling:
The concentration of the Test Item was measured at the applied concentration at the start and at the end of the renewal periods.
Duplicate samples (Set 1 and Se 2) were taken from each concentration at the beginning and at the end of the renewal periods at the applied test concentration level as well as from the Control.
Vehicle:
no
Details on test solutions:
Because the Test Item was very poorly soluble in water (see water solubility study performed in Seibersdorf Laboratories, Citoxlab study code: 17/003-910AN), 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 (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of Test Item into the test medium (ISO Medium) two days before the start of the experiment. This solution was shaken for approximately 24 hours at approximately 30°C (Day -2) and then was equilibrated for approximately 24 hours at approximately 20°C (Day -1). 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 solution was distributed into test vessels prior to introduction of Daphnia.
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 247 mg/L (as CaCO3).
Test temperature:
The test temperature was in the range of 20.3 – 20.7°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 19.8 and 20.8°C.
pH:
The pH was in the range of 7.11 – 7.70.
Dissolved oxygen:
The dissolved oxygen concentration was in the range of 7.5 – 8.9 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 under semi-static conditions.
Details on test conditions:
TEST CONDITIONS
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 – 20.7°C measured in the test vessels. The additionally measured temperature in the climate chamber was between 19.8 and 20.8°C.
Oxygen concentration: 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 7.5 – 8.9 mg/L.
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 7.11 – 7.70.
Light: The test was carried out in 16-hour light and 8-hour dark cycle.
Hardness: The reconstituted water (ISO medium) had a total hardness of 247 mg/L (as CaCO3).

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. 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 can 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.
During the formulation procedure the stock solution was prepared by the similar method described above (3.1.2.) and the test solutions were prepared by appropriate dilution of this stock solution.

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:
NOEC
Effect conc.:
100 other: % saturated solution (based on a 100 mg/L loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 other: % saturated solution (based on a 100 mg/L loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % saturated solution (based on a 100 mg/L loading rate)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
There was no immobilized 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 concentrations [% saturated solution]

Untreated control

0.1

1

10

100

Number of treated / immobilised animals

10 / 0

10 / 0

10 / 0

10 / 0

10 / 0

 

Number of 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% saturated solution (nominal)

20

0

0

0

0

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this Daphnia magna acute immobilisation study the calculated and observed endpoints for the effect of the Test Item were the followings:
The 24h and 48h EC50 value: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)
The 48h EC100 value: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)
The 48h NOEC: 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)
The 48h LOEC: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated.)
Based on the results of this study, the Test Item had no toxic effect at saturation; 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 the Test Item was assessed with Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

 

Toxic effects were not observed during the preliminary concentration range-finding test therefore a limit test was performed in the main experiment using only one test concentration at the limit of solubility of the Test Item in the test medium (100 % saturated solution from a Loading Rate of 100 mg/L) and one control group.

 

The analytical phase of this study was conducted at the Test Site, Seibersdorf Labor GmbH, in Austria.

 

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

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. All validity criteria were met during this study.

 

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

 

The 24h and 48h EC50 value: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

The 48h EC100 value: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

The 48h NOEC: 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

The 48h LOEC: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

 

Based on the results of this study, the Test Item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium. The selenium content of the measured vessels were <0.0081 mg/L in each case.

 

Description of key information

The 24h and 48h EC50 value: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

The 48h EC100 value: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

The 48h NOEC: 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

The 48h LOEC: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated) 

Key value for chemical safety assessment

Additional information

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

Toxic effects were not observed during the preliminary concentration range-finding test therefore a limit test was performed in the main experiment using only one test concentration at the limit of solubility of the Test Item in the test medium (100 % saturated solution from a Loading Rate of 100 mg/L) and one control group.

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

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. All validity criteria were met during this study.

 

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

 

The 24h and 48h EC50 value: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

The 48h EC100 value: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

The 48h NOEC: 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

The 48h LOEC: > 100% saturated solution (Based on a 100 mg/L loading rate; the solution is considered to be saturated)

 

Based on the results of this study, the Test Item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium. The selenium content of the measured vessels were <0.0081 mg/L in each case.