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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:
01 September 2017 to 13 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
(2004)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
At the start of the exposure (0 hours), samples were taken after preparation of the test item concentrations and analyzed. For the test item analysis after 24 hours, separate replicates were prepared at the beginning of the exposure phase without daphnids and incubated under test conditions until sampling.
Samples of the 48-hours old media were taken directly from the test vessels (containing daphnids) at the end of the exposure (48 hours).
All samples were stored at room temperature until the start of analysis, if necessary. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A saturated solution prepared with a nominal loading of 100 mg/L of the test item and 4 subsequent dilution levels in a geometric series with a separation factor of 2.2 were prepared out of the stock solution by dilution with dilution water , were tested as follows:
4.27 - 9.39 - 20.7 - 45.5 - 100 mg/L
A saturated solution with a nominal loading of 100 mg test item/L was prepared with dilution water three days prior to the start of the exposure in a glass flask. An appropriate amount of the test item (density of 0.824 g/cm3 were taken into account) was placed onto the surface of the dilution water by a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 72 ± 1 hour. After completion of stirring followed by a settlement period of 4 hours, the homogeneous phase (i.e. the saturated solution) was taken by siphoning from the approximate center of the water body and used for testing. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). Since no Tyndall effect was observed, the saturated solution was used without any further efforts like centrifugation or prolongation of the settlement period.
- Eluate: Elendt M4 Medium (as per OECD 202
- Differential loading: Not applicable
- Controls: Dilution water without test item incubated under the same conditions as the test groups.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna STRAUS (Clone 5
- Justification for species other than prescribed by test guideline: Not applicable
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age of parental stock (mean and range, SD): Not reported
- Feeding during test: No
- Food type: Not applicable
- Amount: Not applicable
- Frequency: Not applicable

ACCLIMATION
- Acclimation period: In-house culture
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.
- Feeding frequency: The daphnids were fed at least 5 times per week ad libitum
- Health during acclimation (any mortality observed): None reported

QUARANTINE (wild caught)
- Duration: Not applicable
- Health/mortality: Not applicable

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Medium recipe modified to achieve a total water hardness of 160 to 180 mg CaCO3/L
Test temperature:
18 - 22 °C, constant within ± 1 °C
pH:
As per guideline
Dissolved oxygen:
As per guideline
Salinity:
Not applicable
Conductivity:
As per guideline
Nominal and measured concentrations:
Refer to data tables
Details on test conditions:
TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw caps were used.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: A test volume of approximately 130 mL was provided in each test vessel, when filled up to the top with the test solutions without any headspace.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- 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): Not applicable
- Biomass loading rate: 26 mL/daphnid

TEST MEDIUM / WATER PARAMETERS
Same composition as the culture medium

OTHER TEST CONDITIONS
- Adjustment of pH: pH of test/culture medium = 8.2 +/- 0.8
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The EC10-value after 48 hours and the EC100-values (after 24 and 48 hours) were empirically derived from the observation data (lowest concentration level resulting in 100% immobilization).
All effect concentrations (EC10 / 50 / 100) given are based on the time weighted average concentrations of the test item 2-Methyldecan-1-al. Additionally, the effect concentrations are given based on the nominal test item concentrations.

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
31.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: transformation product
Remarks:
2-methyldecanoic acid
Basis for effect:
mobility
Details on results:
All concentration levels were visually clear throughout the exposure period. No Tyndall effect was observed in the saturated solution before insertion of the daphnids.

The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure (0 hours), after 24 hours and in the 48 hours old media at the end of the exposure in all concentration levels and the control.

The measured concentrations of the test item after derivatization were < LOQ of the nominal values at the start of the exposure (0 hours), after 24 hours and at the end of the exposure (48 hours). Therefore, the concentrations of the transformation product 2-methyldecanoic acid were analyzed and calculated as 2-Methyldecan-1-al concentrations by conversion with a factor of 0.914 (based on the ratio of the molar weights of the test item and its transformation product). The time weighted average concentrations of the test item were calculated based on these concentrations to be: 1.56 – 3.68 – 8.11 – 21.3 – 47.5 mg/L.

Additionally, all concentration levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (0 hours), after 24 hours of exposure and the end of the exposure (48 hours).
Results with reference substance (positive control):
- Results with reference substance valid?
- Relevant effect levels: 24h EC50
- Limit test: No
- Dose-response test: Yes
- ECx: EC50 = 1.92 mg/L (95 % CI: 1.11 - 2.00 mg/L)
- Other: Valid range, 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5, 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
Reported statistics and error estimates:
The EC10- and the EC50-value after 24 hours were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism.
The EC50-value after 48 hours was calculated as the geometric mean of the highest concentration level resulting in 0% immobilization and the lowest concentration level causing 100% immobilization, since only one partial effect was observed. Both concentration levels are used as 95% confidence limits. The EC10 after 48 hours is given as a range between both concentration levels without confidence limits. The concentration-effect relationships after 24 and 48 hours are shown graphically.
The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.

Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

2-Methyldecan-1-al

IMMOBILIZATION [%]

Nominal

test item

concentration

[mg/L]

Time weighted

average

concentration

[mg/L]

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

100

47.5

80

60

80

40

65

100

100

100

100

100

 45.5

21.3

 0

 0

 0

 0

 0

   0

   0

   0

   0

   0

 20.7

 8.10

 0

 0

 0

 0

 0

 20

   0

   0

   0

   5

   9.39

 3.68

 0

 0

 0

 0

 0

   0

 20

   0

   0

   5

   4.27

 1.56

 0

 0

 0

 0

 0

   0

   0

   0

   0

   0

Control

 0

 0

 0

 0

 0

   0

   0

   0

   0

   0

 

Measured Concentrations of the Transformation Product calculated as2-Methyldecan-1-alduring the Definitive Test

Sampling

date

Fresh media,

0 hours

Old media,

24 hours

Old media,

48 hours

Time weighted

average

test item

concentration

[mg/L]

Nominal

test item

concentration

[mg/L]

2‑Methyldecan-1-al

Meas.

conc.

[mg/L]

Calc.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

Calc.

conc.

[mg/L]

%

Meas.

conc.

[mg/L]

Calc.

conc.

[mg/L]

%

100

45.9

42.4

42

62.0

57.2

57

39.8

36.8

37

47.5

 45.5

23.3

21.5

47

26.2

24.2

53

17.7

16.3

36

21.3

 20.7

 9.24

 8.53

41

 9.90

 9.14

44

 6.56

 6.05

29

 8.11

   9.39

 4.12

 3.81

41

 4.50

 4.15

44

 3.03

 2.80

30

 3.68

   4.27

 1.68

 1.55

36

 1.95

 1.80

42

 1.30

 1.20

28

 1.56

Control

< LOQ

< LOQ

< LOQ

 

Meas. conc = measured concentration of the transformation product 2-Methyldecanoic acid, dilution factor taken into account

Calc. conc. = equivalent concentration of2‑Methyldecan-1-alcalculated from the measured concentration of the transformation product (conversion factor: 0.914, based on the ratio of molar weights of 2‑Methyldecan‑1‑al:   2-Methyldecanoic acid, 170.3 g/mol : 186.3 g/mol)

% = percentage of the nominal concentration of the test item

LOQMetabolite = limit of quantification of the analytical method for the transformation product 2-methyldecanoic acid (0.3 mg 2-methyldecanoic acid/L)

LOQTest item = limit of quantification of the analytical method for the test item (0.1 mg test item/L)

Water Quality Parameters at the Start of the Exposure (0 hours)

(measured in one additional replicate (without daphnids) per concentration level and control)

2-Methyldecan-1-al

pH-value

Dissolved
O2 concentration

[mg/L]

Nominal

test item

concentration

[mg/L]

Time weighted

average

concentration

[mg/L]

100

47.5

7.30

8.24

 45.5

21.3

7.31

8.58

 20.7

 8.10

7.25

8.64

   9.39

 3.68

7.24

8.61

   4.27

 1.56

7.16

8.62

Control

8.01

9.12

 

 

Water Quality Parameters at the End of the Exposure (48 hours)

(measured in one replicate per concentration level containing daphnids)

2-Methyldecan-1-al

pH-value

Dissolved
O2 concentration

[mg/L]

Replicate number

Nominal

test item

concentration

[mg/L]

Time weighted

average

concentration

[mg/L]

100

47.5

7.17

6.91

1

 45.5

21.3

6.71

4.73

1

 20.7

 8.10

7.15

6.29

1

   9.39

 3.68

7.14

5.55

2

   4.27

 1.56

7.22

6.33

1

Control

7.21

6.40

1

 

 

Water Quality Parameters of the Dilution Waterat the Start of the Exposure (0 hours)

Dilution water

dated:

pH-value

 

 

Dissolved

O2 concentration [mg/L]

Temperature

 

[°C]

Conductivity

 

[µS/cm]

Total hardness

 

[mg CaCO3/L]

2017-10-10

8.01

9.12

20.4

432

175

Validity criteria fulfilled:
yes
Conclusions:
Based on the time weighted average concentrations of the test item 2-Methyldecan-1-al, the 48 hours-EC50 for Daphnia magna was 31.8 mg/L (with confidence limits: 21.3 – 47.5 mg/L).
Based on the nominal concentrations of the test item 2-Methyldecan-1-al, the 48 hours-EC50 for Daphnia magna was 67.5 mg/L (with confidence limits: 45.5 – 100 mg/L).
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined at the test facility according to OECD 202 (2004).

The study was conducted in a closed system (sealed glass flasks) without headspace under static conditions over a period of 48 hours with a saturated solution prepared with a nominal loading of 100 mg/L of the test item and 4 subsequent dilution levels in a geometric series with a separation factor of 2.2. The nominal concentrations of the test item were: 4.27 – 9.39 – 20.7 – 45.5 – 100 mg/L.

All concentration levels were visually clear throughout the exposure period. No Tyndall effect was observed in the saturated solution before insertion of the daphnids.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure (0 hours), after 24 hours and in the 48 hours old media at the end of the exposure in all concentration levels and the control.

The measured concentrations of the test item after derivatization were < LOQ of the nominal values at the start of the exposure (0 hours), after 24 hours of exposure and at the end of the exposure (48 hours). Therefore, the concentrations of the transformation product 2-methyldecanoic acid were analyzed and calculated as 2‑Methyldecan‑1‑al concentrations by conversion with a factor of 0.914 (based on the ratio of the molar weights of the test item and its transformation product). The time weighted average concentrations of the test item were calculated based on these concentrations to be: 1.56 – 3.68 – 8.11 – 21.3 – 47.5 mg/L.

Additionally, all concentration levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (0 hours), after 24 hours of exposure and the end of the exposure (48 hours).

All effect concentrations are based on the time weighted average concentrations of the test item 2-Methyldecan-1-al. Additionally, the effect concentrations are given based on the nominal test item concentrations.

The validity criteria of the test guideline were fulfilled.

Based on the time weighted average concentrations of the test item 2-Methyldecan-1-al, the 48 hours-EC50 for Daphnia magna was 31.8 mg/L (with confidence limits: 21.3 – 47.5 mg/L).

Based on the nominal concentrations of the test item 2-Methyldecan-1-al, the 48 hours-EC50 for Daphnia magna was 67.5 mg/L (with confidence limits: 45.5 – 100 mg/L).

Description of key information

48h EC50 (Daphnia magna) = 31.8 mg/L (meas.), OECD 202; Anon., 2017

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
31.8 mg/L

Additional information

In a single key study, the effects of the test item to Daphnia magna were determined at the test facility according to OECD 202 (2004).

To account for degradation of the parent substance aldehyde to acid or further degradation products, preliminary testing was conducted to assess the stability of the test item under test conditions. After 24 hours of slow stirring, followed by a 1 hour settlement phase, all measured concentrations via LC-MS/MS of test item were below LOQ. A second stability assessment was conducted with analysis of the test item (parent) and the corresponding transformation product (2 -methyldecanoic acid) using a saturated solution with a nominal concentration of 100 mg/L. Slow stirring was applied to the test solution. Analytical samples were taken at 0, 24, 48 and 72 hours after initiation of stirring.

Measured concentrations of the parent substance were <LOQ at all time points. The highest concentration of transformation product was found after 72 hours of stirring. The measured amount of transformation product was calculated as the corresponding amount of parent. This preliminary work was used to design the medium preparation method for this study. In accordance with OECD Series on Testing and Assessment No. 23 (2000), when chemical substances have a very short half-life, it is more relevant to assess the toxicity of the major transformation product(s) as the toxicity of this entity is more relevant for actual environmental exposure. Therefore, the acute toxicity testing on aquatic invertebrates is based on the measured amount of transformation product expressed as the corresponding amount of parent.

The study was conducted in a closed system (sealed glass flasks) without headspace under static conditions over a period of 48 hours with a saturated solution prepared with a nominal loading of 100 mg/L of the test item and 4 subsequent dilution levels in a geometric series with a separation factor of 2.2. The nominal concentrations of the test item were: 4.27 – 9.39 – 20.7 – 45.5 – 100 mg/L.

All concentration levels were visually clear throughout the exposure period. No Tyndall effect was observed in the saturated solution before insertion of the daphnids.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control.

The concentrations of the test item were analytically verified via LC-MS/MS in the fresh media at the start of the exposure (0 hours), after 24 hours and in the 48 hours old media at the end of the exposure in all concentration levels and the control.

The measured concentrations of the test item after derivatization were < LOQ of the nominal values at the start of the exposure (0 hours), after 24 hours of exposure and at the end of the exposure (48 hours). Therefore, the concentrations of the transformation product 2-methyldecanoic acid were analyzed and calculated as 2‑Methyldecan‑1‑al concentrations by conversion with a factor of 0.914 (based on the ratio of the molar weights of the test item and its transformation product). The time weighted average concentrations of the test item were calculated based on these concentrations to be: 1.56 – 3.68 – 8.11 – 21.3 – 47.5 mg/L.

Additionally, all concentration levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (0 hours), after 24 hours of exposure and the end of the exposure (48 hours).

All effect concentrations are based on the time weighted average concentrations of the test item 2-Methyldecan-1-al. Additionally, the effect concentrations are given based on the nominal test item concentrations.

The validity criteria of the test guideline were fulfilled.

Based on the time weighted average concentrations of the test item 2-Methyldecan-1-al, the 48 hours-EC50 for Daphnia magna was 31.8 mg/L (with confidence limits: 21.3 – 47.5 mg/L).

Based on the nominal concentrations of the test item 2-Methyldecan-1-al, the 48 hours-EC50 for Daphnia magna was 67.5 mg/L (with confidence limits: 45.5 – 100 mg/L).

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