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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:
26 February 2017 - 18 March 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:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd 28 October 2016
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and 1.0, 1.8, 3.2, 5.6 and 10% 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 in used solutions 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)
- In order to determine the solubility of the substance under test conditions, a media preparation trial was conducted. No significant difference was observd between stirring (24/48 h) followed by centrifugation at 10000 g for 30 minutes, centrifugation at 40000 g for 30 minutes, filtration with 1 liter discarded and filtration with 2 litres discarded.
- 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, 5.6, 3.2, 1.8 and 1.0% (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 addition of the test item, 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: not specified

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
Hardness:
250 mg/L as CaCO3
Test temperature:
21 °C - 22 °C
pH:
fresh media: 7.5-8.0
used media: 7.6-8.0
Dissolved oxygen:
fresh media: 8.9-9.3 mg O2/L
used media: 8.6-8.9 mg O2/L
Nominal and measured concentrations:
Nominal test concentrations (defintive test): 1.0, 1.8, 3.2, 5.6 and 10% of a saturated solution prepared at a loading rate of 50 mg/L
Measured concentrations: 0.34, 0.63, 1.2, 2.3 and 4.0 mg/L (mean initial measured test concentrations in fresh solutions)

Since there was no significant change in the measured concentrations at 24 and 48 hours (see table 2) the results are based on the mean initial measured test concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL, sealed conical flasks
- Fill volume: 150 mL
- 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: 528-552 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: 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; effects were expected between 1 and 10% of the saturated solution prepared at a loading rate of 50 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (February/March 2016)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.76 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: mean of measured concentrations in freshly pepared solutions because the substance was stable during the renewal period.
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
- Other: Relative to initial [%] ranged from 91-98% in the first renewal period, and 92-100% 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.

ToxRat Professional was used to perform the statistical analyses.

Table 1       Cumulative immobilization data and observations in the range-finding 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

1.8

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

5.6

0

0

0

0

0

0

5 N

5 N

5 N

5 N

10

3

0

0

1

4

20

2 N

5 N

5 N

4 N

N = No sub-lethal effects observed

C = Covered in debris

T = Trapped at surface

A/I = All daphnia immobilized

Table 2 Cumulative Immobilization Data and Observations in the Definitive Test

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

1.0

0

0

1

1

2

10

5 N

5 N

4 N

4 N

1.8

0

0

1

0

1

5

5 N

5 N

4 N

5 N

3.2

5

5

5

5

20

100

A/I

A/I

A/I

A/I

5.6

5

5

5

5

20

100

A/I

A/I

A/I

A/I

10

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

Table 3       Measured test concentrations

Time Point

Nominal Concentration of
Test Item in Test Sample

cnom

Sample Preparation Factor



F

Determined Concentration of Test Item in Test Sample

 


c

[Hours]

[% v/v Saturated Solution]

 

[mg/L]

0

Control

0.1

<LOQ

(fresh)

1.0

0.1

0.353

 

1.8

0.1

0.633

 

3.2

0.1

1.22

 

5.6

0.1

2.36

 

10

0.1

4.11

24

Control

0.1

<LOQ

(old)

1.0

0.1

0.327

 

1.8

0.1

0.616

 

3.2

0.1

1.19

 

5.6

0.1

2.15

 

10

0.1

3.93

24

Control

0.1

<LOQ

(fresh)

1.0

0.1

0.320

 

1.8

0.1

0.633

 

3.2

0.1

1.17

 

5.6

0.1

2.30

 

10

0.1

3.81

48

Control

0.1

<LOQ

(old)

1.0

0.1

0.310

 

1.8

0.1

0.623

 

3.2

0.1

1.17

 

5.6

0.1

2.12

 

10

0.1

3.82

LOQ = Limit Of Quantification

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 0.76 mg/L based on mean initial measured test concentrations in freshly prepared media.
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 a pliminary media preparation trial it was observed that an initial loading rate of 100 mg/L caused over-saturation of the solution (tiny particles/globules of test item were present). Therefore, the initial loading rate was reduced to 50 mg/L, which was eventually chosen as maximum loading rate in the tests. Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, 1.0, 1.8, 3.2, 5.6 and 10% (v/v) dilutions of a saturated solution prepared at loading rate of 50 mg/L. 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.

Results: Measured concentrations in used test solutions ranged from 91 -98% of initial in the first renewal period, and from 92 -100% of initial in the second renewal period. Since test concentrations remained stable during the 24 -hour renewal periods, results were based on the measured concentrations in the freshly prepared media. The mean initial measured concentrations were: 0.34, 0.63, 1.2, 2.3 and 4.0 mg/L. All validity criteria were fulfilled and the study was considered to be valid. The 48h-EC50 value to Daphnia magna was 0.76 mg/L based on mean initial 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.In a pliminary media preparation trial it was observed that an initial loading rate of 100 mg/L caused over-saturation of the solution (tiny particles/globules of test item were present). Therefore, the initial loading rate was reduced to 50 mg/L, which was eventually chosen as maximum loading rate in the tests. Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, 1.0, 1.8, 3.2, 5.6 and 10% (v/v) dilutions of a saturated solution prepared at loading rate of 50 mg/L. 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 periodswere analysed with a validated GC-FID method.

Results: Measured concentrations in used test solutions ranged from 91 -98% of initial in the first renewal period, and from 92 -100% of initial in the second renewal period. Since test concentrations remained stable during the 24 -hour renewal periods, results were based on the measured concentrations in the freshly prepared media. The mean initial measured concentrations were:0.34, 0.63, 1.2, 2.3 and 4.0 mg/L. All validity criteria were fulfilled and the study was considered to be valid. The 48h-EC50 value to Daphnia magnawas 0.76 mg/L based on mean initial measured concentrations.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.76 mg/L

Additional information