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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 February 2017 - 09 April 2017
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:
2004
Deviations:
yes
Remarks:
spacing factor of nominal test solution series was > 2.2 (actual factor: 3.1-3.2)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
yes
Remarks:
spacing factor of nominal test solution series was > 2.2 (actual factor: 3.1-3.2)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and all test groups
- Sampling method: 100 mL samples were taken from the freshly prepared bulk test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of a saturated solution (direct addition to test medium) - A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water (Elendt M7) 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 stock solutions of 1.0, 3.2, 10 and 32% (v/v) saturated solution.
- Controls: test medium without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
- Other: No information on aeration of the test medium.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory culture
- Feeding during culture:
* Food type: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
* Frequency: daily
- Feeding during test: no
- Age at study initiation: young daphnids with an age of < 24 hours

ACCLIMATION
- Acclimation period: no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult daphnia were maintained in 150 mL glass beakers containing 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. 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.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
none
Hardness:
250 mg/L as CaCO3
Test temperature:
19 °C - 22 °C
pH:
fresh media: 7.8
used media: 7.7-8.0
Dissolved oxygen:
fresh media: 8.7-9.3 mg O2/L
used media: 8.0-8.8 mg O2/L
Nominal and measured concentrations:
Nominal test concentrations (defintive test): 1.0, 3.2, 10, 32 and 100% (v/v) of a saturated solution prepared at a loading rate of 100 mg/L
Mean measured initial concentrations:0.34, 0.98, 3.1, 9.7 and 31 mg/L corresponding with 1.0, 3.2, 10, 32 and 100% (v/v) of the saturated solution

Considering that no significant change in measured concentrations was observed in used test solutions at 24 and 48 h, mean measured initial test concentrations were used to calculate effect parameters.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass stoppered conical flasks
- Fill volume: approximately 100 mL, or maybe completely filled (unclear from the report; in any case sealed to reduce evaporation)
- Aeration: no
- Renewal rate of test solution: yes, after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 medium according to OECD 202
- Culture medium different from test medium: no
- Intervals of water quality measurement: water temperature, pH and dissolved oxygen concentrations were recorded daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 528 to 571 Lux
- Other: for renewal at 24 hours, the daphnids were transferred by wide bore pipette from the 24-Hour old test media into the fresh test media

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility (at 24 and 48 hours)

RANGE-FINDING STUDY
- Test concentrations RF: 0.10, 1.0, 10 and 100% v/v saturated solution (measured concentration in 100% saturated solution was ca. 19 mg/L)
- Test concentrations initial test: 0.10, 0.32, 1.0 and 10% (v/v) of a saturated solution prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (test conducted between 20 October 2016 and 22 October 2016)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
5.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
Concentration remained stable (> 80%)
Basis for effect:
mobility
Remarks on result:
other: 95% C.I. 3.1-9.7 mg/L
Details on results:
- Observed sub-lethal effects: no
- Mortality of control: no
- Other adverse effects control: no
- Effect concentrations exceeding solubility of substance in test medium: no

Individual pH and dissolved oxygen values remained within acceptable limits throughout the duration of the study.
Temperature was recorded at 19 – 22 °C, exceeding the maximum deviation of ± 1 °C. This was considered not to have affected the validity and integrity of the study as this deviation did not coincide with any mortalities or sub lethal effects.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Test concentrations: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- 48h-EC50: 1.2 mg/L (95% C.I. 1.1-1.3 mg/L)
- Other: Analysis of the immobilization data was carried out using the Trimmed Spearman-Karber method at 48 hours (ToxRat Professional).
Reported statistics and error estimates:
The EC50 values and associated confidence limits at 24 and 48 hours were calculated by Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Verification of test concentrations

Initial measured concentrations in freshly prepared solutions at t=0 and t=24 h were in the range 28.6 - 37.6% of nominal loading rates (at 1.0, 3.2, 10, 32 and 100% (v/v) of the saturated solution prepared at a loading rate of 100 mg/L). Relative to initial [%] for the first and second renewal period were calculated by the reviewer.

Based on the results, it was considered justified to base effect parameters on mean measured initial concentrations.

Table 1 - Measured concentrations in test samples

Time Point

Nominal Concentration of
Test Item in Test Sample

cnom

 

Sample Preparation Factor

F

Determined Concentration of Test Item in Test Sample

 


c

 

Relative to initial

 

[Hours]

[% v/v Saturated Solution]

 

[mg/L]

[%]

0

Control

0.10

<LOQ

 

1.0

0.10

0.376

 

 

3.2

0.10

1.07

 

 

10

0.10

3.10

 

 

32

0.10

9.69

 

 

100

0.10

31.1

 

 

 

 

 

 

24

Control

0.10

<LOQ

 

(old)

1.0

0.10

0.336

89

 

3.2

0.10

0.997

93

 

10

0.10

3.03

98

 

32

0.10

9.81

101

 

100

0.10

30.1

97

 

 

 

 

 

 24*

Control

0.10

<LOQ

 

(fresh)

1.0

0.10

0.295

 

 

3.2

0.10

3.08

 

 

10

0.10

0.888

 

 

 

 

 

 

48

Control

0.10

<LOQ

 

(old)

1.0

0.10

0.267

91

 

3.2

0.10

0.889

100

 

10

0.10

2.75

89

LOQ = Limit of Quantification

* Samples taken for analysis at 24 hours (fresh media) for the 3.2 and 10% v/v saturated solution test concentrations appeared to be switched. Given that the 48-Hour samples show that the vessels were dosed correctly it is considered justifiable to report the original 24-Hour fresh results as follows; the 3.2% v/v saturated solution as 0.888 mg/L and the 10% v/v saturated solution as 3.08 mg/L.

Mean measured initial concentrations were 0.34, 0.98, 3.1, 9.7 and 31 mg/L.

Biological effects

Table 2            Cumulative Immobilization Data and Observations in the Definitive Test

Nominal
Concentration
(% v/v Saturated Solution)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

1.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

3.2

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

5

5

5

5

20

100

A/I

A/I

A/I

A/I

100

5

5

5

5

20

100

A/I

A/I

A/I

A/I

 

Nominal
Concentration
(% v/v Saturated Solution)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

1

0

0

0

1

5

4 N

5 N

5 N

5 N

1.0

0

0

0

0

0

0

5 N

5 N

5 N

5 N

3.2

1

0

0

0

1

5

4 N

5 N

5 N

5 N

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

5

5

5

5

20

100

A/I

A/I

A/I

A/I

100

5

5

5

5

20

100

A/I

A/I

A/I

A/I

R1– R4= Replicates 1 to 4

N = No sub-lethal effects observed

A/I = All daphnia immobilized

Validity criteria fulfilled:
yes
Conclusions:
The 48h-EC50 value to Daphnia magna was 5.5 mg/L (OECD TG 202) which are based on mean measured concentrations in freshly prepared test solutions because the substance is stable over 24 hours (concentrations initial > 80%).
Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions. In the definitive test, the following dose levels were tested: untreated control, 1.0, 3.2, 10 and 32 % (v/v) dilutions of a saturated solution prepared at loading rate of 100 mg/L, as well as the 100% saturated solution. For each concentration and the control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours closed vessels. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24 -hour renewal periods were analysed with a validated GC-FID method. Mean measured concentrations in freshly prepared test solutions were 0.34, 0.98, 3.1, 9.7 and 31 mg/L corresponding with 1.0, 3.2, 10, 32 and 100% (v/v) of the saturated solution. Measured concentrations in used test solutions ranged from 89 -101% of initial in the first renewal period, and from 89 -100% of initial in the second renewal period. Since test concentrations remained stable during the 24 -hour renewal periods (i.e. within 80 -120% of initial), mean measured initial concentrations were used to derive effect parameters because the concentrations remained stable. After 24 h, all daphnids in the two highest test solutions were found to be immobilized, whereas no sub-lethal effects were observed in the control replicates and test solution replicates up to 10% (v/v) of the saturated solution. Based on this only the lowest three test solutions were renewed and daphnids observed for mobility after 48 hours. The 48h-EC50 value to Daphnia magna was 5.5 mg/L based on mean measured concentrations in freshly prepared test solutions.

Description of key information

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions. In the definitive test, the following dose levels were tested: untreated control, 1.0, 3.2, 10 and 32 % (v/v) dilutions of a saturated solution prepared at loading rate of 100 mg/L, as well as the 100% saturated solution. For each concentration and the control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours closed vessels. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24 -hour renewal periods were analysed with a validated GC-FID method. Mean measured concentrations in freshly prepared test solutions were 0.34, 0.98, 3.1, 9.7 and 31 mg/L corresponding with 1.0, 3.2, 10, 32 and 100% (v/v) of the saturated solution. Measured concentrations in used test solutions ranged from 89 -101% of initial in the first renewal period, and from 89 -100% of initial in the second renewal period. Since test concentrations remained stable during the 24 -hour renewal periods (i.e. within 80 -120% of initial), mean measured initial concentrations were used to derive effect parameters because the concentrations remained stable. After 24 h, all daphnids in the two highest test solutions were found to be immobilized, whereas no sub-lethal effects were observed in the control replicates and test solution replicates up to 10% (v/v) of the saturated solution. Based on this only the lowest three test solutions were renewed and daphnids observed for mobility after 48 hours. The 48h-EC50 value to Daphnia magna was 5.5 mg/L based on mean measured concentrations in freshly prepared test solutions.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
5.5 mg/L

Additional information