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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:
from 2017-05-08 to 2017-10-19
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
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
high performance liquid chromatography with UV detection
Details on sampling:
- Concentrations: 6.25, 12.5, 25.0, 50.0, 100.0 mg/L
- Sampling method: duplicate samples were taken from each test concentration and the control at the start and at the end of the test (after 48 hours). For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: Immediately after sampling, acetonitrile (10 mL acetonitrile per 10 mL sample volume) was added to each sample to stabilize the latter during the storage period. Thereafter, all samples were frozen (at -20 ±5 °C). Based on pre-experiments for investigation of the storage stability (non-GLP IES Study 20170154), the test item was found to be stable in the test water under these storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Stock solution: The highest nominal concentration of 100 mg/L was prepared by completely dissolving 108.16 mg of test item in 1080 mL of test water using ultrasonic treatment for 2 minutes and intense stirring for 15 minutes at room temperature in a closed flask
Test solutions: This highest test concentration was diluted with test water to prepare the test media of the lower test concentrations
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna Straus
- Source: Daphnia Collection of the University of Basel/Switzerland in 2015
- Age: 6-24 hours old, not first brood progeny
- Feeding during test: The daphnids were not fed during the test.

ACCLIMATION
- Acclimation conditions: The cultivation of the parental daphnids is performed in reconstituted water of the quality identical to the water quality used in the tests (with respect to pH, main ions, and total hardness).
- Type and amount of food: During breeding, daphnids were generally fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA GmbH, 49324 Melle / Germany).

Test type:
static
Water media type:
freshwater
Remarks:
Reconstituted test water (ISO Test water) according to OECD Guideline No. 202
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
20 - 21 °C
pH:
7.6 - 7.8
Dissolved oxygen:
8.1 - 8.6 mg/L
Nominal and measured concentrations:
0 h:
nominal: 25, 50, 100 mg/L
determined: 23.6, 48.2, 99.0 mg/L

48 h:
nominal: 25, 50, 100 mg/L
determined: 22.4, 44.2, 90.8 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass tubes
- Type: closed
- Material, size, headspace, fill volume: glass, no headspace, 60 mL fill volume
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water
- Ca/Mg ratio: 4:1
- Alkalinity: 0.8

OTHER TEST CONDITIONS
- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used.
- Light intensity: 15 - 17 μmol m-2 s-1.

EFFECT PARAMETERS MEASURED: The immobility of the daphnids was determined by visual inspection after 24 and 48 hours of exposure. Those daphnids not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized. Observations were also performed for other non-lethal effects (e.g. abnormal behavior or appearance).

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
85 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
71 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: none
- Any observations (e.g. precipitation): Test solutions were clear throughout the entire test duration
Results with reference substance (positive control):
EC50 (24 h) = 1.2 mg/L (March 2017)
Reported statistics and error estimates:
The 48-hour EC50 and the 95% confidence limits were calculated by Probit Analysis using linear maximum likelihood regression
The 48-hour NOEC, EC0 and EC100 were determined directly from the raw data.

Effect of the test substance on the Mobility of Daphnia magna

Nominal

Test Substance Concentration [mg/L]

No. of

Daphnids

Tested

Immobilized

Daphnids after

24 Hours

Immobilized

Daphnids after

48 Hours

No

[%]

No

[%]

Control 6.25

20

0

0

0

0

12.5

20

0

0

0

0

25

20

0

0

0

0

50

20

0

0

3

15

100

20

13 (7F)

65

17 (3F)

85

F: slower swimming activity

Analytical Results for Test Samples

Sampling Day/ Sample Age

Nominal Concentration of
Test Item
cnom

Measured Concentration
 of Test Item

x

Sample Preparation Factor

F

Determined Concentration of Test Item

c

% of Nominal Concentration

[d/h]

[mg/L]

[mg/L]

[mg/L]

[%]

0/0

Control

n.d.

2

< LOQ

n.a.

(fresh)

25

11.8

2

23.6

94

 

50

24.1

2

48.2

96

 

100

49.5

2

99.0

99

2/48

Control

n.d.

2

< LOQ

n.a.

(aged)

25

11.2

2

22.4

90

 

50

22.1

2

44.2

88

 

100

45.4

2

90.8

91

Results for Samples from Volatility Test

Sampling

Day/ Sample

Age

Nominal

Concentration

of

Test Item

Measured

Concentration

of Test Item

Sample

Preparation

Factor

Determined

Concentration

of Test Item

% of Nominal

Concentration

[d/h]

Cnom [mg/L]

X [mg/L]

F

c[mg/L]

[%]

0/0 (fresh)

100

45.7

2

91.4

91

100

45.0

2

90.1

90

3/72 (aged, open system)

100

7.2

2

14.4

14

100

7.06

2

14.1

14

3/72 (aged, closed system)

100

44.3

2

88.6

89

100

43.9

2

87.9

88

Validity criteria fulfilled:
yes
Conclusions:
In an acute Daphnia immobilisation test according to OECD Guideline 202, an EC50 (48 h) of 71 mg/L was determined.
Executive summary:

The acute toxicity of the test item on Daphnia magna was determined in a 48-hour static test according to the OECD Guideline 202.The nominal test item concentrations tested were 6.25, 12.5, 25, 50 and 100 mg/L. Additionally, a control (test water without test substance) was tested in parallel. As the test item is a volatile substance, the test was performed using glass tubes completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system). The measured concentrations of the test substance in the test media of the test concentrations from 25 to 100 mg/L were between 94 and 99 % of the nominal values at the start of the test and between 88 and 91 % at the end of the test. Thus, the correct dosage of the test substance was confirmed. The test item was stable in the test media over the test period of 48 hours. Therefore, the endpoint values were related on the nominal concentrations of the test item. The samples from the nominal test concentrations of 6.25 and 12.5 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results.

The 48 -hour EC50 was calculated to be 71 mg/L. The 48 -hour EC0 and NOEC of the test substance were both 25 mg/L, since no toxic effect was observed up to and including this test concentration.

Description of key information

In an acute Daphnia immobilisation test according to OECD Guideline 202, a EC50 (48 h) of 71 mg/L was determined (2017).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
71 mg/L

Additional information

The acute toxicity of the test item on Daphnia magna was determined in a 48-hour static test according to the OECD Guideline 202 (2017).The nominal test item concentrations tested were 6.25, 12.5, 25, 50 and 100 mg/L. Additionally, a control (test water without test substance) was tested in parallel. As the test item is a volatile substance, the test was performed using glass tubes completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system). The measured concentrations of the test substance in the test media of the test concentrations from 25 to 100 mg/L were between 94 and 99 % of the nominal values at the start of the test and between 88 and 91 % at the end of the test. Thus, the correct dosage of the test substance was confirmed. The test item was stable in the test media over the test period of 48 hours. Therefore, the endpoint values were related on the nominal concentrations of the test item. The samples from the nominal test concentrations of 6.25 and 12.5 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results.

The 48-hour EC50 was calculated to be 71 mg/L. The 48-hour EC0 and NOEC of the test substance were both 25 mg/L, since no toxic effect was observed up to and including this test concentration.