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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:
20 June 2016 - 14 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD series on testing and assessment number 23
Version / remarks:
December 14, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and 10, 18, 32, 56 and 100% of a saturated solution
- Sampling method: samples (100 mL) were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the pooled replicates at 24 and 48 hours.
- Sample storage conditions before analysis: test samples were analyzed on the day of receipt
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 (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 10, 18, 32 and 56% (v/v) saturated solution.
- Controls: test medium without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no - At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
- Other: Prior to use the test medium was aerated until the dissolved oxygen concentration was approximately air-saturation value.
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:
21 °C - 22 °C
pH:
fresh media: 7.7-7.9
used media: 7.7-8.1
Dissolved oxygen:
fresh media: 8.6-9.1 mg O2/L
used media: 8.5-8.8 mg O2/L
Nominal and measured concentrations:
Based on the results of the range-finding test, performed at nominal test concentration of 0.10, 1.0 10 and 100% (v/v) of a saturated solution (measured concentration in 100% saturated solution was ca. 3.3 mg/L) the following test concentrations were assigned to the definitive test:

Nominal test concentrations (defintive test): 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 50 mg/L
Measured concentrations: 0.10, 0.10, 0.24, 0.38 and 0.70 mg/L (geometric mean measured concentration)

Geometric mean measured test concentrations were used to calculate effect parameters in order to give a “worst case” analysis of the data.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass stoppered conical flasks
- Fill volume: approximately 100 mL (in principle no headspace as test vessels were completely filled and sealed so as to reduce losses due to volatilization)
- 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: ISO medium according to OECD 202
- Culture medium different from test medium: yes - culture medium: Elendt M7
- Intervals of water quality measurement: water temperature, pH and dissolved oxygen concentrations were recorded daily

OTHER TEST CONDITIONS
- Adjustment of pH: yes; where needed, pH was adjusted to pH 7.8+/- 0.2
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 464 to 527 Lux
- Other: for renewal at 24 hours, the daphnids 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: 0.10, 1.0, 10 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (March 2016)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% CI 0.21 - >0.80
Details on results:
- Observed sub-lethal effects: reduced movement was observed in the 100% v/v saturated solution test concentration at 24 hours:
- Mortality of control: no
- Other adverse effects control: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Other: Relative to initial [%] ranged from 65 - 130% in the first renewal period, and 95-121% in the second renewal period (calculated by the reviewer).

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.
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: 0.64 mg/L
Reported statistics and error estimates:
The EC50 value at 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression.
The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Step-down Cochran-Armitage Test Procedure.

ToxRat Professional was used to perform the statistical analyses.

Table 1. 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

10

0

0

0

0

0

0

5 N

5 N

5 N

5 N

18

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

0

0

0

0

0

0

5 N

5 N

5 N

5 N

56

1

0

0

0

1

5

4 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 R

5 R

5 R

5 R

 

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

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

18

0

0

0

0

0

0

5 N

5 N

5 N

5 N

32

0

1

0

0

1

5

5 N

4 N

5 N

5 N

56

1

0

1

1

3

15

4 N

5 N

4 N

4 N

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

R = Reduced movement

A/I = All daphnia immobilized

 

Verification of Test Concentrations

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours (see Table 3 below) measured test concentrations to range from less than the LOQ to 0.76 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed that measured test concentrations ranged from less than the LOQ to 0.73 mg/L, hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

The geometric mean measured test concentrations were determined to be:

 

Table 2. Geometric mean measured test concentrations

Nominal Test Concentration (% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

10

0.10

18

0.10

32

0.24

56

0.38

100

0.70

Table 3. Test sample results

Time Point

Nominal Concentration of Test Item in Test Sample

Sample Preparation Factor

Determined Concentration of Test Item in Test Sample

 cnom

F

c

[hours]

[% v/v Saturated Solution]

[mg/L]

0 (Fresh)

Control

0.1

<LOQ

10

0.1

0.0695

18

0.1

0.173

32

0.1

0.279

56

0.1

0.465

100

0.1

0.756

24 (Old)

Control

0.1

0.0681 (<LOQ)

10

0.1

0.0905

18

0.1

0.112

32

0.1

0.227

56

0.1

0.305

100

0.1

0.659

24 (Fresh)

Control

0.1

<LOQ

10

0.1

0.0748

18

0.1

0.129

32

0.1

0.242

56

0.1

0.398

100

0.1

0.665

48 (Old)

Control

0.1

0.0587 (<LOQ)

10

0.1

<LOQ

18

0.1

0.156

32

0.1

0.229

56

0.1

0.378

100

0.1

0.728

LOQ = Limit of Quantification = 0.20 mg/L.

LOQ discussion: Recoveries were carried out at 0.1 mg/L, which corresponded to the concentration obtained for the 10% v/v saturated solution. The recoveries were repeated twice and gave an 89% recovery rate with an RSD of 15% (n = 5) and a 69% recovery rate with an RSD of 18% (n = 5). Due to the co-elution of peaks at the retention time of the test item, the recoveries were repeated at 0.2 mg/L and gave a recovery rate of 94% with an RSD of 3.0% (n = 5). The LOQ was therefore assessed as 0.20 mg/L. Sample concentrations at approximately 0.1 mg/L are recoverable, but with a greater degree of imprecision, and are reported for indicative purposes only.

Validity criteria fulfilled:
yes
Remarks:
1) In the control, no daphnids became immobilised or showed other signs of disease or stress. 2) The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Conclusions:
The 48h-EC50 value to Daphnia magna was 0.45 mg/L based on geometric mean measured concentrations.
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. Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, 10, 18, 32 and 56% (v/v) dilutions of a saturated solution prepared at loading rate of 50 mg/L, as well as the 100% saturated solution. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight 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. Measured concentrations in used test solutions ranged from 65 -130% of initial in the first renewal period, and from 95 -121% of initial in the second renewal period. Since test concentrations did not remain stable during the 24 -hour renewal periods (i.e. not within 80 -120% of initial), geometric mean measured concentrations were calculated. The range tested based on geometric mean measured concentrations was 0.10, 0.10, 0.24, 0.38 and 0.70 mg/L. The 48h-EC50 value to Daphnia magna was 0.45 mg/L based on geometric mean measured concentrations.

Description of key information

A study was performed to assess the acute toxicity of the substance toDaphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions.Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, 10, 18, 32 and 56% (v/v) dilutions of a saturated solution prepared at loading rate of 50 mg/L, as well as the 100% saturated solution.For each concentration and a control group, twentyDaphnia magna(less than 24 hours old) were exposed for 48 hoursin air-tight 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 atthe start and the end of both 24 -hour renewal periodswere analysed with a validated GC-FID method.Measured concentrations in used test solutions ranged from 65 -130% of initial in the first renewal period, and from 95 -121% of initial in the second renewal period. Sincetest concentrations did not remain stable during the 24 -hour renewal periods (i.e. not within 80 -120% of initial), geometricmean measured concentrations were calculated. The range tested based on geometric mean measured concentrations was 0.10, 0.10, 0.24, 0.38 and 0.70 mg/L.The 48h-EC50 value toDaphnia magnawas 0.45 mg/L based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.45 mg/L

Additional information