Reaction mass of 5,9-Undecadienal-2,6,10-trimethyl-, (5E)- and 5,9-Undecadienal-2,6,10-trimethyl-, (5Z)-

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:

18 - 23 Sep 2016

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)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations and controls were analytically verified
- Sampling method: samples were taken at the start of exposure from newly prepared test concentrations; at the end of exopsure intervals (24 and 48 h) samples were taken directly from test vessels
- Sample storage conditions before analysis: stored at room temperature before preparation

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: test item placed onto water surface; bottle was closed with the screw cap; gentle stirring for 72 h with a magnetic stirrer at room temperature; saturated solution was collected by siphoning through a glass tube; saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with ISO test water
- Controls: yes, test medium control

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: 2-24 h
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, light intensity of max. 1500 lux; culture medium: Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.
- Feeding during test: none
- Food type (for stock culture): with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: with an algae cell density of > 10 E+06 cells/mL
- Frequency: 5 times per week ad libitum

ACCLIMATION
- Acclimation period: at least 2 h
- Acclimation conditions: same as test

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: removal of juvenile daphnids at the latest 22 h before test start and discarded. The juveniles born within the following period of max. 22 h preceding the exposure were used for the test after an acclimatization phase of at least 2 h in the dilution water. No first brood progeny was used for the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

267 mg CaCO3/L

Test temperature:

20.5 - 20.8 °C (start of exposure intervals 0 and 24 h)

pH:

7.64 - 7.69 (control, start of exposure intervals 0 and 24 h)
6.99 - 7.05 (Test item concentrations, start of exposure intervals 0 and 24 h)
7.06 - 7.36 (control, end of exposure interval 24 h)
7.19 - 7.62 (Test item concentrations, end of exposure intervals 24 h)
7.40 - 7.54 (Control, end of exposure interval 48 h)
7.57 - 7.62 (Test item concentrations, end of exposure intervals 48 h)

Dissolved oxygen:

8.06 - 8.21 (control, start of exposure intervals 0 and 24 h)
5.16 - 7.80 (test item concentration, start of exposure intervals 0 and 24 h)
7.58 - 7.97 (control, end of exposure interval 24 h)
5.43 - 7.84 (test item concentration, end of exposure interval, 24 h)
5.22 - 7.56 (control, end of exposure interval 48 h)
5.12 - 7.40 (test item concentration, end of exposure interval, 48 h)

Conductivity:

653 - 654 µS/cm (start of exposure Intervals 0 and 24 h)

Nominal and measured concentrations:

control, 6.25, 12.5, 25.0, 50.0 and 100% of the saturated solution (nominal)
control, 117, 238, 745, 941 and 1755 µg/L (based on the geometric mean measured concentration)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: closed, sealed glass flasks with screw caps
- Material, size, headspace: glass, size: 4.5 (IC) x 9.5 (H) cm, ca. 130 mL, headspace: nearly no headspace, 130 mL test volume for each test vessel
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): every 24 h (freshly prepared test solutions were filled in a second set of test vessels and the daphnids were transferred with a pipette)
- 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: Culture Medium Elendt M4 according to guideline

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 lux

EFFECT PARAMETERS MEASURED: Immobilization (observed every 24 h)

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY:
- Test concentrations: control, 1, 10 and 100% saturated solution
- Results used to determine the conditions for the definitive study: Based on the results of the non-GLP range-finding test, the main test was performed with the following concentration range: 6.25, 12.5, 25.0, 50.0 and 100% (saturated solution) correspond to 117, 238, 745, 941 and 1755 µg/L (based on the geometric mean of each test item concentration).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.127 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits: 0.117 - 0.222 mg/L

Details on results:

- Mortality of control: no daphnids were immobilized or showed any signs of disease or stress
- Other: Biological and analytical results are summarized within the tables 1-3 in the section "Any other information on results incl. tables"

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 (24 h): 2.03 mg/L

Reported statistics and error estimates:

The ECxx-values (after 24 and 48 hours) were estimated empirically from the observation data. All effect levels were based on the geometric mean measured concentrations the test item. The ECxx-value after 24 and 48 hours was calculated by sigmoidal dose-response regression with the software GraphPad Prism5. 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 Prism5. Calculations were made using the following software: GraphPad Prism5, GraphPad Software, Inc. and Excel, Microsoft Corporation

Table 1: Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

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

Test item		IMMOBILIZATION [%]
Dilution level of the saturated solution [%]	Geometric mean measured test item concentration [µg/L]	24 hours					48 hours
		Replicates					Replicates
		1	2	3	4	MV	1		2	3	4		MV
100*	1755*	100	100	100	100	100	100% mortality after 24 hours
50.0	941	100	100	100	100	100
25.0	745	100	100	100	100	100
12.5	238	100	100	100	60	90	100	100		100	100	100
6.25	117	0	0	0	0	0	20	0		0	0	5
Control	0	0	0	0	0	0	0	0		0	0
* = saturated   solution

Table 2: Measured Concentrations of the Test Item during the Definitive Test

Sampling date	2016-09-21 Start of the exposure interval 0 hours	2016-09-22 End of the exposure interval 24 hours		2016-09-22 Start of the exposure interval 24 hours	2016-09-23 End of the exposure interval 48 hours		Geometric mean measured test item concentration [µg/L]
Start of analysis	2016-09-21 1)	2016-09-22 1)		2016-09-22 1)	2016-09-23
Dilution level of the Saturated solution [%]	Test item
	Meas.conc. [µg/L]	Meas.conc. [µg/L]	%	Meas.conc. [µg/L]	Meas.conc. [µg/L]	%
100*	1844	1671	91	21512)	Not determined due to 100% mortality after 24 hours		1755
50.0	1152	769	67	Not determined due to 100% mortality after 24 hours			941
25.0	622	893	144				745
12.5	306	194	63	266	204	77	238
6.25	159	100	63	145	81.6	56	117
Control	< LOQ	< LOQ		< LOQ	< LOQ

Meas. conc. = measured concentration of the test item, mean value of 2 injections, dilution factors taken into account

% = percent of the initially measured concentrations of the test item at the start of the respective exposure interval

LOQ = limit of quantification of the analytical method (3 µg/L of the test item)

* = saturated solution

1) = analyzed on 2016-09-23

2) = not used for the calculation of the geometric mean measured concentration. Only analyzed to show the right preparation.

Table 2: Measured Concentrations of the Test Item at Glass Walls of Test Vessels during the Definitive Test

Test Item
Dilution level of the saturated solution [%]	2016-09-21 Start of the exposure interval 0 hours	2016-09-22 End of the exposure interval 24 hours		2016-09-22 Start of the exposure interval 24 hours	2016-09-23 End of the exposure interval 48 hours
	Measured initial concentration	Adsorbed to glass		Measured initial concentration	Adsorbed to glass
	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%
100*	1844	41.8 1)	2	Not determined, due to 100% mortality after 24 hours
12.5	Not determined			145	< LOQ

Meas. conc. = measured concentration of the test item, mean value of 2 injections, extraction factors taken into account

% = percent of the initially measured concentrations of the test item at the start of the respective exposure interval

LOQ = limit of quantification of the analytical method (3 µg/L of the test item)

* = saturated solution

1) = reanalyzed on 2016-09-23

Validity criteria fulfilled:

yes

Conclusions:

The test substance caused immobilisation of Daphnia magna in a guideline study according to OECD 202 (GLP). An EC50 (48 h) of 0.127 mg/L based on the geometric mean measured concentration was calculated.

Description of key information

EC50 (48 h) = 0.127 mg/L (Daphnia magna, OECD 202, based on geometric mean measured concentration)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.127 mg/L

Additional information

One experimental study is available investigating the short-term effects of the substance to aquatic invertebrates. The study was performed according to the OECD 202 (GLP) with the water flea Daphnia magna. A saturated solution was tested with the following dilution series of 6.25, 12.5, 25.0, 50.0 and 100% of the saturated solution (nominal; 117, 238, 745, 941, 1755 µg/L based on geometric mean measured concentrations) under semi-static conditions. It was ensured by an appropriate test design that the substance does not volatilize from the test vessels (closed vessels; no headspace). The concentrations of the test item were analytically verified via LC-MS/MS at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all tested concentration levels and the control. The measured concentrations of the saturated solution were 1844 µg/L at the start of the exposure (0 hours) and 2151 µg/L at renewal (24 hours).At the end of the exposure intervals (24 and 48 hours), the measured concentrations were within the range of 56 to 144% of the initially measured concentrations.Based on these results the endpoints were evaluated using the geometric mean measured concentrations. Immobilisation of D. magna was observed after 48 h resulting in an EC50 (48 h) of 127 µg/L (0.127 mg/L) based on geometric mean measured concentrations.