(E)-dodec-2-en-1-al

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in accordance with international guidelines (OECD TG 202) and in accordance with GLP. The study design was modified in accordance with OECD 23 (2000) and ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b. The study met all relevant validity criteria.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

In addition to compliance with OECD TG 202 and EU Method C.2., modifications to the testing approach were conducted in accordance with OECD 23 (2000) and ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b. The aqueous half-life of the test item was determined to <48h in aqueous testing medium, so it was concluded that the test should be conducted on the transformation products rather than the parent substance (see "Details on Test Solutions"); as environmental exposure will be to the transformation products.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

Before the start of the exposure phase, the saturated solution was the test item analytically verified (double determination) at 0, 8, 24, 48, 72, 96, 164, 192 and 216 hours until the test item concentration was below the LOQ in both replicates. Before the start of the exposure phase, the samples for determination were taken for all measurement points (see above) after siphoning of the saturated solution and stored in completely filled and tightly closed vials at room temperature until the start of analysis. At the start of the exposure (0 hours), sampling was carried out after preparation of the loading levels. At the end of the exposure (48 hours), samples were taken directly from the test vessels.
Additionally a sample of the saturated solution after siphoning (0 hours) and all loading levels and the control during the exposure phase (at 0 and 48 hours) were analytically verified via analysis of total carbon (TOC, according to DIN EN 1484).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A saturated solution with a nominal loading of 10.0 mg/L of the test item was prepared in duplicate with dilution water (see below) eleven days before the start of the exposure phase. 11.5 mg of the test item were weighed out and transferred with 1150 mL of the dilution water (ISO test water as specified below) into a glass flask with a side-arm (nominal capacity 1 L, actual capacity approx.. 1150 mL), which was then completely filled. The stock solution was stirred with approximately 1100 rpm for 24 hours at 30 °C. During the stirring period, hydrolysis of the test item was expected. The stock solution had nearly no headspace during the stirring period to reduce losses of the test item by evaporation. After completion of stirring, the stock solution was allowed to stand for 24 hours to cool down at room temperature and for separation of undissolved test item. The saturated solution was removed by side-arm of the flask (from the approximate bottom of the glass flask). Due to the density of the test item, any undissolved material was expected to float on the surface. The two replicates of the saturated solution were pooled and stored in a completely filled and tightly closed glass flask under test conditions until the start of the exposure. To ensure complete hydrolysis, the test item concentration in the saturated solution was measured in duplicate during storage at regular intervals (e.g. at 0, 8 and 24 hours and then until the test item concentration was below the LOQ). Since the test item concentration was below the LOQ after 9 days of storage, the dilution levels were prepared and the daphnids were inserted. Samples for the determination of complete transformation of the test item in the saturated solution were taken for all measurement points after siphoning of the saturated solution and stored in completely filled and tightly closed vials at room temperature until the start of analysis.
The saturated solution was used as highest test loading level and as stock solution for preparation of further loading levels by diluting with dilution water. The saturated solution was checked for a Tyndall effect with a laser pointer beforehand. No Tyndall effect was observed at the saturated solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System: Daphnia magna STRAUS (Clone 5)
- Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany - Breeder: Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Culture Conditions: 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. 20 µEm-2 s-1 (max. 1340 lx)
- Culture Medium: Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was used.
- Culture Feewding: The culture daphnids were fed 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 > 106 cells/mL. The algae were cultured at the test facility.
- Origin of Food Algae: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

Hardness of the diltion water at test initiation was 260 mg CaCO3/L

Test temperature:

During the test period, the temperature in the incubator was 19 - 20 °C.

pH:

Refer to Tables 5 and 6.

Dissolved oxygen:

Refer to Tables 5 and 6.

Salinity:

Conductivity of the dilution water at test initiation was 656 µS/cm.

Nominal and measured concentrations:

5 loading levels of the test item in a geometric series with a separation factor of 2.2, prepared by diluting the saturated solution with dilution water, were tested as follows: 4.27 - 9.39 - 20.7 - 45.5 - 100% of the saturated solution. The loading rates were selected ibased on the results of a non GLP preliminary range finding test. Results of analytical monitoring are presented in Table 4.

Details on test conditions:

Test Vessels: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw caps (made from polypropylene) were used. A test volume of approximately 130 mL was provided in each test vessel.
Dilution Water: ISO test water; according to OECD 202, Annex 3.
Number of Daphnids and Replicates: 20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each loading level and the control.
Age of the Daphnids at the Start of Exposure: 2 to 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels latest 22 hours before the start of the exposure and discarded. The juveniles born within this period of max. 22 hours preceding the exposure were used for the test after an acclimatisation phase of at least 2 hours in the dilution water. No first brood progeny was used for the test.
Acclimation: At least 2 hours in dilution water.
Application: The test vessels were filled up with the test solutions. The daphnids were inserted with a small amount of dilution water by pipette. Thereafter, the test vessels were closed immediately with screw caps.
Test temperature (target): 18 - 22°C, constant within ± 1°C
Illumination (target): Diffuse light, light intensity of max. 20 µEm-2 s-1 (max. 1340 lx)
Photoperiod (target): 16/8 hours light/dark cycle
Feeding: The daphnids were not fed during the study.

Reference substance (positive control):

yes

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.76 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Transformation products

Basis for effect:

other: Immobilisation

Remarks on result:

other: 95% CL: 3.84 – 5.94 mg/L

Details on results:

Measured concentrations of the test item reduced to below the LOQ after a storage period of 216 hours, then the exposure phase was started.
Biological Data in the Definitive Test:
The percentage immobility, determined in all test and control groups after 24 and 48 hours, is given in Table 1. The study was performed under static conditions in a closed system.
Additional Observations during the Definitive Test:
The tested solutions were visually clear throughout the exposure period and no Tyndall effect was observed at the saturated solution.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 hours. The EC50-value with 95% confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was: EC50: 1.93 mg/L (CI 1.14 - 3.24 mg/L) The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Reported statistics and error estimates:

The EL50-value after 48 hours was estimated by probit analysis with the software ToxRat, Version 3.2.1. The 95% confidence limits were calculated. The EL50-value after 24 hours was calculated as the geometric mean of the lowest loading level resulting in 100% immobilisation (10.0 mg/L) and the highest loading level resulting in 0% immobilisation (4.55 mg/L), since only the highest loading level resulted in 100% immobilisation and all other loading levels showed 0% immobilisation. Thus, the two highest loading levels will be used as 95% confidence limits. The EC50-value for the reference item was calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations for the reference item were carried out from the best-fit values with the software GraphPad Prism5.

Table 1. Immobilisation in Daphnia after 24 hours and 48 hours Exposure in the Definitive Test

		IMMOBILISATION [%]
Dilution level of the saturated solution [%]	Nominal loading level of the test item [mg/L]	24 hours			48 hours
		Replicates			Replicates
		1	2	MV	1	2	MV
100*	10.0	100	100	100	100	100	100
45.5	4.55	0	0	0	40	20	30
20.7	2.07	0	0	0	10	10	10
9.39	0.939	0	0	0	0	0	0
4.27	0.427	0	0	0	0	0	0
Control		0	0	0	0	0	0

* Saturated Solution

Table 2. Measured Concentrations of the Test Item in the Saturated Solution During the Storage Period

Saturated solution of the Test Item (2E)‑2‑Dodecenal
Start of analysis	Storage period   [hours]	Mean measured* test item concentration [µg/L]
2016-02-08	0	2129
2016-02-09	8	2083
2016-02-09	24	1450
2016-02-10	48	730
2016-02-11	72	320
2016-02-12	96	303
2016-02-15	164	65.6
2016-02-16	192	23.3
2016-02-17	216	< LOQ

* Mean value of 2 replicates with 2 injections each, dilution factor taken into account

LOQ limit of quantification of the analytical method (15µg/L)

Table 3. Measured TOC Concentrations during the Definitive Test

Sampling date	2016-02-08 Start of the storage phase, 0 hours (before complete hydrolysis)	2016-02-17 Start of the exposure, 0 hours (after complete hydrolysis)	2015-02-19 End of the exposure, 48 hours (after complete hydrolysis)
Start of analysis	08/02/2016	17/02/2016	19/02/2015
Nominal loading level of the test item (mg/L)	Total Organic Carbon (TOC)
	Measured concentration (mg C/L)]^1)	Measured concentration (mg C/L)]^1)	Measured concentration (mg C/L)]^1)
10	13.87	11.12	9.93
5	Not determined	6.79	6.5
2.5		4.61	5.06
1.25		3.86	4.48
0.625		3.69	5.56
Control		< 2.00	< 2.00

1) measured TOX concentration, mean value of 2 injections

Table 4. Measured Concentrations of the Test Item during Definitive Test

Sampling date	2016-02-17 Start of the exposure, 0 hours	2016-02-19 End of the exposure, 48 hours
Start of analysis	2016-02-17	2016-02-19
Dilution level of the saturated solution [%]	(2E)‑2‑Dodecenal
	Meas. conc. [µg/L]	Meas. conc. [µg/L]
100*	< LOQ	< LOQ
45.5	< LOQ	< LOQ
20.7	< LOQ	< LOQ
9.39	< LOQ	< LOQ
4.27	< LOQ	< LOQ
Control	< LOQ	< LOQ

* Saturated Solution

Meas. Conc. measured concentration of the test item, mean value of 2 injections, dilution factor taken into account

LOQ limit of quantification of the analytical method (15µg/L)

Table 5. Water Quality Parameters at the Start of the Exposure

Nominal loading level of the test item (mg/L)	pH	Dissolved O2 Concentration (mg/L)
10	7.36	5.49
4.55	7.67	8.18
2.07	7.7	8.63
0.939	7.67	8.79
0.427	7.63	8.55
Control	7.99	9.18

Table 6. Water Quality Parameters at the End of the Exposure (48 hours)

Nominal loading level of the test item (mg/L)	pH		Dissolved Oxygen Concentration (mg/L)
	Replicate 1	Replicate 2	Replicate 1	Replicate 2
10	7.29	7.26	3.98	4.07
4.55	7.42	7.39	6.15	6.38
2.07	7.42	7.43	6.81	7.15
0.939	7.54	7.50	7.90	8.01
0.427	7.48	7.53	7.64	8.07
Control	7.42	7.44	7.95	7.86

Statistics

Parameters of the probit analysis with immobility at 48 h: Results of the regression analysis

Parameter                      Value

Computation runs:           8

Slope b:                          4.79812

Intercept a:                     -3.25275

Variance of b:                 0.91151

Goodness of Fit Chi²:     5.16744

Degrees of freedom:       3

p(Chi²):                           0.160

Log LC50:                       0.67792

SE Log LC50:                  0.04462

g-Criterion:                      0.15209

F:                                  14.663

p(F) (df: 1;3):                  0.031

 

Chi² is a goodness of fit measure. If the probability, p(Chi²), is lower or equal than 0.100 data is much scattering round the computed dose/response function. In this case and with quantal data, confidence limits are corrected for heterogeneity (extra-binomial variance).

 

A statistically significant concentration/response was found (p(F) <= 0.05; i.e. slope of the relationship is significantly different from zero).

 

Results of the probit analysis with immobility at 48 h: Selected effective concentrations (LCx) of the test item and their 95%-confidence limits (according to Fieller`s theorem).

 

  Toxicity Metric          LC10            LC20           LC50

      Value [mg/L]         2.575           3.181           4.763

      lower 95%-cl         1.623           2.232           3.835

     upper 95%-cl         3.291           3.936           5.940

n.d.: not determined due to mathematical reasons or inappropriate data

 

Slope function after Litchfield and Wilcoxon: 1.616 

 

(The slope function is derived from the slope, b, of the linearized probit function and computes as S = 10^(1/b); please note that small values refer to a steep concentration/response relation and large ones to a flat relation.)

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal loading levels of the test item, the 48 hour EC50 of the test item transformation products to Daphnia magna was 4.76 mg/L (95% confidence limits: 3.84 – 5.94 mg/L).

Executive summary:

In an acute immobilisation test with Daphnia magna (STRAUS), the effects of the degradation products after complete transformation of the test item were determined according to OECD TG 202 (2004), which is equivalent to Council Regulation (EC) No. 440/2008 Method C.2 (2008). The study was conducted under static conditions over a period of 48 hours with a saturated solution with a nominal loading of 10.0 mg/Lof the test item and further four dilution levels in a geometric series with a separation factor of 2.2 (nominal loading rates of 4.27 to 45.5%). A saturated solution with a nominal loading of 10.0 mg/Lof the test item was prepared in duplicate with dilution water (ISO test water, acc. to OECD TG 202, Annex 3) eleven days before the start of the exposure period and was used as highest loading level and as stock solution for the preparation of all further tested dilution levels. To ensure complete hydrolysis, the saturated solution was stored under test conditions in a completely filled and closed glass flask and the concentration of the test item in the saturated solution was measured in duplicates at regular intervals (i.e. at 0, 8, 24, 48, 72, 96, 168, 192, 216 hours). After 192 hours, the mean measured concentration of the test item was 23.3 µg/L which is close to the LOQ of 15 µg/L. The measured test item concentration in both measured replicates was below the LOQ after a storage period of 216 hours, the Daphnids were inserted. Twenty Daphnids, divided into 2 replicates with ten Daphnids each, were exposed to each loading level and the control. The tested solutions were visually clear throughout the exposure period. The concentrations of the test item were analytically verified via LC-MS/MS during the storage period and at the start (0 hours) and at the end of the exposure phase (48 hours) in all concentration levels and the control. During the storage phase the mean measured test item concentration in the saturated solution were as follows: 2129 µg/L (0 hours), 2083 µg/L (8 hours), 1450 µg/L (24 hours), 730 µg/L (48 hours), 320 µg/L (72 hours), 303 µg/L (96 hours), 65.6 µg/L (164 hours), 23.3 µg/L (192 hours) and < LOQ (216 hours). The measured concentrations of the test item at the start (0 hours) and the end of the exposure phase (48 hours) were below the LOQ (15 µg/L). Additionally, the total carbon concentration (TOC, according to DIN EN 1484) of the saturated solution after siphoning (start of the storage phase, 0 hours) and the TOC in all loading levels and in the control at the start (0 hours) and at the end of the exposure (48 hours) was analytically verified. Since the test item was completely transformed and the measured test item concentrations were all below the LOQ, the effect values are based on the nominal loadings of the test item. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 hours) and at the end of the exposure (48 hours), were within the acceptable limits. The validity criteria of the test guidelines were fulfilled. Based on the nominal loading levels of the test item, the 48 hour EC50 of the test item transformation products to Daphnia magna was 4.76 mg/L (95% confidence limits: 3.84 – 5.94 mg/L).

Description of key information

48-h EC50 = 4.76 mg/L (transformation products); OECD TG 202 (modified to OECD 23), 2016

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.76 mg/L

Additional information

Key data: OECD TG 202, 2016 : In an acute immobilisation test with Daphnia magna (STRAUS), the effects of the degradation products after complete transformation of the test item were determined according to OECD 202 (2004), which is equivalent to Council Regulation (EC) No. 440/2008 Method C.2 (2008). The study was conducted under static conditions over a period of 48 hours with a saturated solution with a nominal loading of10.0 mg/L of the test item and further four dilution levels in a geometric series with a separation factor of2.2(nominal loading rates of4.27 to 45.5%). A saturated solution with a nominal loading of 10.0 mg/L of the test item was prepared in duplicate with dilution water (ISO test water, acc. to OECD 202, Annex 3) eleven days before the start of the exposure period and was used as highest loading level and as stock solution for the preparation of all further tested dilution levels. To ensure complete hydrolysis, the saturated solution was stored under test conditions in a completely filled and closed glass flask and the concentration of the test item in the saturated solution was measured in duplicates at regular intervals (i.e. at 0, 8, 24, 48, 72, 96, 168, 192, 216 hours). After 192 hours, the mean measured concentration of the test item was 23.3 µg/L which is close to the LOQ of 15 µg/L. The measured test item concentration in both measured replicates was below the LOQ after a storage period of 216 hours, the Daphnids were inserted. Twenty Daphnids, divided into 2 replicates with ten Daphnids each, were exposed to each loading level and the control. The tested solutions were visually clear throughout the exposure period. The concentrations of the test item were analytically verified via LC-MS/MS during the storage period and at the start (0 hours) and at the end of the exposure phase (48 hours) in all concentration levels and the control. During the storage phase the mean measured test item concentration in the saturated solution were as follows: 2129 µg/L (0 hours), 2083 µg/L (8 hours), 1450 µg/L (24 hours), 730 µg/L (48 hours), 320 µg/L (72 hours), 303 µg/L (96 hours), 65.6 µg/L (164 hours), 23.3 µg/L (192 hours) and < LOQ (216 hours). The measured concentrations of the test item at the start (0 hours) and the end of the exposure phase (48 hours) were below the LOQ (15 µg/L). Additionally, the total carbon concentration (TOC, according to DIN EN 1484) of the saturated solution after siphoning (start of the storage phase, 0 hours) and the TOC in all loading levels and in the control at the start (0 hours) and at the end of the exposure (48 hours) was analytically verified. Since the test item was completely transformed and the measured test item concentrations were all below the LOQ, the effect values are based on the nominal loadings of the test item. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 hours) and at the end of the exposure (48 hours), were within the acceptable limits. The validity criteria of the test guidelines were fulfilled. Based on the nominal loading levels of the test item, the 48 hour EC50 of the test item transformation products to Daphnia magna was 4.76 mg/L (95% confidence limits: 3.84 – 5.94 mg/L).