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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:
02 Feb - 29 Feb 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
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. certificate)
Remarks:
Federal Office for the Environment (FOEN), Switzerland
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 2.2, 4.6 and 10 mg/L (0.46 and 1 mg/L were not analysed since the concentrations were below the NOEC)
- Sampling method: Duplicate samples were taken at the start and end of each medium renewal period. For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: Samples were stored deep-frozen at about -20 °C immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 100 mg/L (nominal) was prepared in a completely filled and tightly closed stirring vessel by dissolving 55 mg (day 0) and 55.19 mg (day 1) of the test item in 550 mL of test water. This solution was stirred for 15 min at room temperature. This stock solution was used to prepare the 10 mg/L test solution. The lower concentrations were prepared by serial dilution of the highest test concentration. Test media were prepared just before the introduction of daphnids.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: in-house culture since 2015 (originally obtained from Daphnia Collection of the University of Basel/Switzerland)
- Age at study initiation (mean and range, SD): < 24 h
- Method of breeding: Kept under same conditions as test. Daphnids were fed three times a week with an algal suspension of Desmodesmus subspicatus (Strain: 86.81 SAG) or a commercial fish diet Tetra Min® (Tetra Werke, Melle, Germany)
- Feeding during test: none
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg CaCO3/L
Test temperature:
21 °C
pH:
7.8 - 8.0
Dissolved oxygen:
8.6 - 9.1 mg O2/L
Nominal and measured concentrations:
nominal: 0.46, 1.0, 2.2, 4.6 and 10 mg/L
measured: see "Any other information on results incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass tubes, completely filled with medium to avoid losses.
- Type: closed
- Aeration: no
- Renewal rate of test solution: every 24 h
- 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: according to guideline
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was monitored continuously; pH and O2 concentration were measured at the beginning and end as well as at medium renewal after 24 d of the old and new medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark (20 min transition period)
- Light intensity: 15-18 µmol m-2 s-1

EFFECT PARAMETERS MEASURED
- Mobility after 24 and 48 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.17
- Range finding study
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: 100% mortality were observed at the concentration of 10 and 100 mg/L whereas no mortality was recorded at 1 mg/L after 48 h.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CI: 2.2 - 4.6 mg/L
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: test solutions were colorless throughout the test
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50/LC50: EC50 (24 h) 0.78 mg/L
Reported statistics and error estimates:
Since the EC50 could not be calculated by Probit analysis or Moving Average Interpolation due to the steep concentration-effect relationship. Instead, the EC50 was determined as the geometric mean value of the two consecutive test concentrations with 0% and 100% immobility (EC0 and EC100).

Table 1: Immobility of D. magna after 24 and 48 h

 

 

Immobilized Daphnids after 24 h [%]

Immobilized Daphnids after 48 h [%]

Nominal test concentration [mg/L]

No of Daphnids tested

No.

%

No.

%

Control

20

0

0

0

0

0.46

20

0

0

0

0

1.0

20

0

0

0

0

2.2

20

0

0

0

0

4.6

20

0

0

20

100

10

20

20

100

20

100

 

Table 2: Measured concentrations during the study

Sampling day/Sampling age [d/h]

Nominal concentration [mg/L]

Determined concentration [mg/L]

% of nominal

0/0 (fresh)

Control

< LOQ

n.a.

2.2

1.90

86

4.6

4.33

94

10

9.00

90

1/24 (aged)

Control

< LOQ

n.a.

2.2

2.06

94

4.6

4.06

88

10

9.03

90

1/0 (fresh)

Control

< LOQ

n.a.

2.2

2.03

92

4.6

4.28

93

2/24 (aged)

Control

< LOQ

n.a.

2.2

2.06

94

4.6

4.27

93

 LOQ: Limit of quantification

The measured concentrations in the test media of the test concentrations of 2.2 to 10 mg/L were between 86 and 94% of the nominal values at the start of the 24-hour exposure periods and between 88 and 94% at the end of these exposure periods.

Thus, the effect concentrations were based on nominal concentrations.

Validity criteria fulfilled:
yes
Conclusions:
The test item had acute toxic effects on Daphnia magna in a 48-hour semistatic test. The 48-hour EC50was calculated to be 3.2 mg/L with 95%-confidence limits of 2.2
and 4.6 mg/L.
Executive summary:

The acute toxicity of the test substance to Daphnia magna was determined in a 48-hour semi-static test according to OECD 202 (GLP). The nominal test item concentrations tested were 0.46, 1.0, 2.2, 4.6 and 10 mg/L.

As the test substance is a semi volatile substance, the test was performed in a closed system. The suitability of the test system was confirmed by HPLC/UV analysis of the test concentrations.

The measured concentrations in the test media were between 86 and 94% of the nominal values at the start of the 24-hour exposure periods and between 88 and 94% at the end of these exposure periods.

Thus, the test substance was stable in the test media over the 24-hour exposure period and the biological results were related to the nominal concentrations.

The test substance had acute toxic effects on the mobility of Daphnia magna; the 48-hour EC50 was calculated to be 3.2 mg/L.

Description of key information

EC50 (48 h): 3.2 mg/L based on immobilisation of Daphnia magna (OECD 202)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
3.2 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic invertebrates. The study was conducted according to OECD 202 (GLP) under semi-static conditions using the water flea Daphnia magna. Test concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg/L were chosen based on the results from a preliminary range-finding test. It was ensured by an appropriate test design that the substance does not volatilize from the test vessels. The suitability of the test system was confirmed by HPLC/UV analysis of the test concentrations. The measured concentrations were ≥88% of the nominal values at the end of the test. Thus, the effect concentrations were based on the nominal values. Inhibition of immobilisation was recorded resulting in an EC50 (48 h) of 3.2 mg/L.