Hexanamide, N-(2-ethylhexyl)-3,5,5-trimethyl-

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-01 to 2014-02-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
The saturated solution prepared with a nominal loading of 2 mL/L of the test item and further four dilution levels prepared with dilution water out of the saturated solution in a geometric series with a separation factor of 2, were tested as follows:
6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution. This corresponds to the geometric mean measured concentrations:
85.4 -173 - 341 - 643 - 1427 µg/L.
The test concentrations were selected based on the results of a non GLP preliminary range finding test under static conditions.

- Sampling method:
All concentration levels and the control were analytically verified via UPLC-MS/MS at the start (0 h) and at the end of the exposure
(48 h). At the start of the exposure (0 h), sampling was carried out immediately after preparation of the test concentrations.
At the end of the exposure (48 h), samples were taken directly from the test vessels.


- Sample storage conditions before analysis: All original samples were stored at room temperature until preparation. Prepared samples were stored in an autosampler 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 with a nominal loading of 2 mL test item/L was prepared with dilution water three days prior to the start of the exposure phase (0 h). The test item was placed by pipetting (10 mL / 5 L) on the surface of the dilution water.
A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at approximately 23 °C. After separation of the phases, after at least 1 hour of standing, the saturated solution was taken from the homogeneous phase and was used as stock solution.
The saturated solution prepared with a nominal loading of 2 mL/L of the test item and further four dilution levels prepared with dilution water out of the saturated solution in a geometric series with a separation factor of 2, were tested as follows:
6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution. This corresponds to the geometric mean measured concentrations:
85.4 -173 - 341 - 643 - 1427 µg/L.

- Dilution water : ISO Test water, according to OECD 202, Annex 3, composition see below

Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123

- Controls: Dilution water without test item tested under the same conditions as the test goups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation: 2 to 24 h
- Method of breeding:
In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2°C, in an incubator, 16 h illumination, light intensity of max. 20 µExm-2 x s-1;
Culture feeding: at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus with an algae cell density of > 10^6 cells/mL.
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L

- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: at least 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

DILUTION WATER:

Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

Dilution water at test start: 262 (mg CaCO3/L)

Test temperature:

At test start: 20.1°C (Water)

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, loosely covered with a glass watch
- Material, size, headspace, fill volume:
Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): Static test (no water renewal)
- 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): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see above
- Conductivity: Dilution water at test start: 659 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and at test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Immobilisation was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Range finding study:
Immobilisation Rates in the non GLP Preliminary Range Finding Test
(n = 20, divided into 2 replicates with 10 daphnids each)

Dilution level of
the saturated solution
of the test item
[%] IMMOBILISATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 50 50 50 70 80 75
10 0 10 5 10 10 10
1 0 0 0 0 0 0
Control 0 0 0 0 0 0


MV = mean value

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

341 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

341 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

475 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.66 (CI: 1.42 - 1.90) mg/L

Reported statistics and error estimates:

The EC100-values after 24 and 48 h and the NOEC and LOEC after 48 h were deduced directly from the observation data, without mathematical calculation.

EC10- and EC50-values after 48 h were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5. The concentration effect relationship is shown graphically. The EC50-value for the reference item and its confidence limits were calculated accordingly.
The EC10- and EC50-values after 24 h could not be calculated due to the lack of effects and were therefore deduced directly from the observation data, without mathematical calculation.

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item the 48 h-EC50 for Daphnia magna was 475 µg/L (95 % confidence limits: 408 – 545 µg/L). The NOEC after 48 h was 341 µg/L. The LOEC after 48 h was 643 µg/L.

Executive summary:

In the acute immobilisation test withDaphnia magna (STRAUS), the effects of the test item were determined according to OECD 202 (2004) from 2014-02-01 to 2014-02-06, with the definitive exposure phase from 2014-02-04 to 2014-02-06.

The test item is yellowish, clear and viscous liquid with a water solubility of 0.5 mg/L and a density of 0.87 g/cm³(20 °C, DIN 51757).

A saturated solution* with a nominal loading of 2 mL test item/L was prepared with dilution water three days prior to the start of the exposure phase (0 h). The test item was placed by pipetting (10 mL / 5 L) on the surface of the dilution water. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with magnetic stirrer at approximately 23 °C. After separation of the phases, after at least 1 hour of standing, the saturated solution was taken from the homogeneous phase and was used as stock solution.

The study was conducted under static conditions over a period of 48 h with the saturated solution with a nominal loading of 2 mL test item/L and further four dilution levels in the range of 6.25 to 100 % of the saturated solution. The dilution levels were prepared with dilution water out of the saturated solution in a geometric series with a separation factor of 2. Twenty daphnids were exposed to each test concentration and the control.

The concentrations of the test item wereanalytically verified via UPLC-MS/MS at the start of the exposure (0 h) and at the end of the exposure (48 h) in all concentration levels and the control.

The initially measured concentrations of the test item at the start of the exposure (0 h) were 106 to 1432 µg/L. At the end of the exposure (48 h), the measured concentrations of the test item were in the range of 65 to 99 % of the initially measured concentrations. The geometric mean measured test item concentrations were calculated to be: 85.4 – 173 – 341 – 643 – 1427 µg/L.

The effect levels given are based on the geometric mean measured concentrations of the test item are given in the table below.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 h) and at the end of the exposure (48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Based onthe geometric mean measured concentrationsof the test item

the 48 h-EC₅₀forDaphnia magnawas 475 µg/L (95 % confidence limits: 408 – 545 µg/L).

 

The NOEC after 48 h was 341 µg/L.The LOEC after 48 h was 643 µg/L.

EC₁₀-, EC₅₀-, EC₁₀₀-Values (with Confidence Interval), NOEC and LOEC

(based on the geometric mean measured concentrations of the test item)

Effect levels	Test duration [h]	test item
		Geometric mean measured concentration [µg/L]	Confidence limits p = 95% [µg/L]
EC10	24	1427	Not applicable
	48	341	< 85.4 – 445
EC50	24	> 1427	Not applicable
	48	475	408 – 545
EC100	24	> 1427	Not applicable
	48	1427	592 – > 1427
NOEC	48	341	Not applicable
LOEC	48	643	Not applicable

Description of key information

Based on the geometric mean measured concentrations of the test item the 48 h-EC50 for Daphnia magna was 475 µg/L (95 % confidence limits: 408 – 545 µg/L). The NOEC after 48 h was 341 µg/L. The LOEC after 48 h was 643 µg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

475 µg/L