dec-1-en-4-yne

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:

25-09-2017 to 29-09-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

An Acute Immobilisation Test to Daphnia magna STRAUS was carried out to determine the EC50-values of the test item after 24 and 48 hours of exposure under semi-static conditions in a closed system

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2016 ; signature: 2017

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All relevant concentration levels and the control were analytically verified via GC-MS in fresh media at the start of exposure and at renewal of the test solutions (0 and 24 hours) and in 24-hours old media at renewal and at the end of the exposure intervals (24 and 48 hours).
- Sampling method: At the start of the exposure and at renewal intervals (0 and 24 hours), sampling was carried out after preparation of the test concentrations. At renewal the end of the exposure intervals (24 and 48 hours), samples of the 24-hour old were taken directly from the test vessels. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C, until analysis if necessary. 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: Test item exposures was prepared from stock solution. A saturated solution with a nominal loading of 100 µL/L of the test item was prepared in duplicate one day prior to the start of the exposure and the renewal of the test solutions (at minus 24 and 0 hours) in tightly closed glass flasks with a lateral outlet . The glass flask (total volume ca. 1150 mL) was filled up to the top with approximately 1150 mL of the dilution water and closed with a screw cap. The test item (115 µL) was placed by pipette under the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 24 hours with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 2 hours for separation of undissolved test item. Thereafter, the saturated solution was removed by the lateral outlet of the flask (from the approximate bottom of the glass flask). The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed. 5 test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25%, 50% and 100% of saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.146, 0.242, 0.534, 1.05 and 2.00 mg/L which were based on analysis during the definitive test period.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: 1.0, 2.0 and 4.0 mg/L were prepared in a separately conducted reference test (documented in the full study report). A negative/blank control without test item or reference item was also included.
- 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.): Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna STRAUS (Clone 5)
- Justification for species other than prescribed by test guideline: Not applicable.
- Age at study initiation (mean and range, SD): < 24 hours old daphnids from a healthy stock were used for the study
- Weight at study initiation (mean and range, SD): Not applicable.
- Length at study initiation (length definition, mean, range and SD): Not applicable.
- Stage and instar at study initiation: Juvenile ; < 24 hours
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not applicable.
- Peripheral shell growth removed prior to test initiation: Not applicable.
- Method of breeding: Not reported. Although the breeder and culture conditions are given in the full study report.
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): Not applicable.
- Feeding during test: No. The daphnids were not fed during the study. During culture: The culture daphnids are 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 > 10^6 cells/mL. The algae are cultured at the test facility
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: At least 2 hours in dilution water.
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): No.

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

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Not applicable.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

In accordance with the OECD TG 202 guideline.

Hardness:

Dilution water quality parameters: 0 hours: Total Hardness: 167 mg CaCO3/L ; 24 hours Total Hardness: 167 mg CaCO3/L

Test temperature:

Dilution water quality parameters: 0 hours: Temperature 20.9 °C ; 24 hours: Temperature 20.9 °C

pH:

0 hours: pH 7.82; 24 hours: pH 8.55 ; mean pH at 48 hours all replicates: did not differ by more than 1.5 units

Dissolved oxygen:

Dilution water quality parameters: 0 hours: O2 dissolved: 8.84 mg/L ; 24 hours: O2 dissolved: 8.85 mg/L; dissolved oxygen at 24 and 48 hours all replicates: did not decrease to less than 3 mg/L.

Nominal and measured concentrations:

Range finding tests were performed (non-GLP) prior to the definitive test: 0 (control), 1%, 10% and 100% saturated solutions.
Definitive test: dilution level of saturated solutions: 0 (control), 6.25%, 12.5%, 25%, 50% and 100%
equivalent Geometric mean measured concentrations were determined: 0 (control), 0.146, 0.242, 0.534, 1.05 and 2.00 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 4.5 (ID) x 9.5 (H) cm, ca. 130 mL
- Type: A semi-static test design with a daily renewal of the test solutions in glass flasks sealed with screw caps (made from polypropylene).
- Material, size, headspace, fill volume: Sealed glass flasks with screw caps (made from polypropylene). Fill volume ca. 130 mL.
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel (divided into 4 replicates).
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water / Elendt M4 according to OECD 202
- Culture medium different from test medium: No. Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L

The test vessels were filled up with the test solutions. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette. Thereafter, the test vessels were closed immediately with screw caps. The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels were filled with the freshly prepared test solutions and the daphnids were transferred by pipette.

OTHER TEST CONDITIONS
- Adjustment of pH: No. The test vessels were not pH adjusted during or after test item exposure.
- Photoperiod: 16/8 h light/dark cycle during culture conditions. During testing: Light exclusion.
- Light intensity: Diffuse light, light intensity of max. 1500 lx during testing.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality), 24 hours and at 48 hours. An organism 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. Other observations were not made.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: 0 (control), 1%, 10% and 100% saturated solutions. See table 1.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 0.867 - 1.45 mg/L ; geometric mean measured concentration

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.564 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 0.392 - 0.848 mg/L ; geometric mean measured concentration

Details on results:

- Behavioural abnormalities: None reported.
- Observations on body length and weight: Not applicable.
- Other biological observations: None reported.
- Mortality of control: No mortalities in control.
- Other adverse effects control: None reported.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Prior to start of the exposure intervals, the test solutions were checked for undissolved test item via laser beam (Tyndall effect). Presence of undissolved test item during preparation and during the test was not observed.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- EC50/LC50: 24h-EC50 was 1.76 mg/L (C.I. 1.54 – 2.01 mg/L).
- Other: 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) listed in DIN 38412 - L 30 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 TG 202

Reported statistics and error estimates:

The EC50-value after 24 and 48 hours of exposure were calculated by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution.

Table 1. Immobilisation Rates in the non GLP Preliminary Range Finding Test

(n = 20, divided into 2 replicates with 10 daphnids each)

Dilution level of saturated solution [%]	Immobilisation [%]
	24 hours				48 hours
	Semi-Static			Static	Semi-Static			Static
	Replicates				Replicates
	1	2	MV	3	1	2	MV	3
100*	100	80	90	80	100	100	100	100
10	0	0	0	0	0	0	5	10
1	0	0	0	0	0	0	0	0
0.01	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0

* saturated solution

 

Table 2. Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

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

Dilution level of saturated solution [%]	Geometric mean measured test item concentrations [mg/L]	Immobilisation [%]
		24 hours					48 hours
		Replicates					Replicates
		1	2	3	4	MV	1	2	3	4	MV
100	2.00	100	100	100	100	100	100*	-	-	-	-
50	1.05	40	40	40	60	85	100	100	100	100	100
25	0.534	40	0	60	0	25	60	20	60	20	40
12.5	0.242	0	0	0	0	0	20	0	0	0	5
6.25	0.146	0	0	0	0	0	0	0	0	0	0
Control		0	0	0	0	0	0	0	0	0	0

* 100% mortality after 24 hours

 

Table 3. Measured Concentrations and Percent of the Initially Measured Concentrations of the Test Item during the Definitive Test

Sampling	0 hours Start of the exposure interval (fresh media)		24 hours End of the exposure interval (old media)		24 hours Start of the exposure interval (fresh media)		48 hours End of the exposure interval (old media)		Geometric mean measured test item concentration [mg/L]
Dilution level of saturated solution [%]	Test item concentration
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
100	2.50	-	1.60	64	2.50 #2	-	ND #1	ND	2.00
50	1.28	-	0.842	66	1.33	-	0.859	64	1.05
25	0.614	-	0.395	64	0.66	-	0.502	75	0.534
12.5	0.301	-	0.192	64	0.337	-	0.175	52	0.242
6.25	0.154	-	0.114	74	0.188	-	0.173	74	0.146
Control	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ

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

% = percent of the initial measured concentration, oat the start of the corresponding exposure interval

LOQ = limit of quantification of the analytical method (0.002 mg/L of the test item or 2µg/L)

#1 Not determined due to 100% mortality

#2 medium was prepared but not used further due to 100% mortality

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was 0.564 mg/L (C.I: 0.392 – 0.848 mg/L) based on geometric mean measured concentrations.

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours with five concentration levels of the test item in the 6.25% to 100% saturated solution concentration range: prepared in a geometric series with a separation factor of 2. To reduce losses of the test item, the study was conducted under semi-static conditions in a closed system. A stock solution of 100% saturation was prepared with modified-Elendt M4 (according to OECD 202, Annex 3) with total hardness of 160 to 180 CaCO3/L with a nominal loading of 100 µL/L of the test item in duplicate one day prior to the start of the exposure and the renewal of the test solutions (at minus 24 and 0 hours) in tightly closed glass flasks with a lateral outlet. The glass flask (total volume ca. 1150 mL) were filled up to the top with approximately 1150 mL of the dilution water and closed with a screw cap. The test item (115 µL) was placed by pipette under the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 24 hours with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 2 hours for separation of undissolved test item. Thereafter, the saturated solution was removed by the lateral outlet of the flask (from the approximate bottom of the glass flask). Five test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25%, 50% and 100% of saturated solution. All test concentrations were visually clear throughout the exposure intervals. Twenty daphnids were exposed to each concentration level and the control. The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette. The concentrations of the test item were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all relevant concentration levels and the control. The measured concentrations of the test item were in the range of 64 to 74% of the 0 hour measured concentrations at 24 hours and 64 to 75% of the 24 hour fresh test solution measured concentrations at 48 hours. The geometric mean measured concentrations were calculated to be: 0.146, 0.242, 0.534, 1.05 and 2.00 mg/L. Since the measured test item concentrations did not remain within ± 20% of the nominal concentrations, the EC50-values are based upon the geometric mean measured concentrations. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EC50 was 0.564 mg/L (C.I: 0.362 – 0.848 mg/L) based on geometric mean measured concentrations of the test item.

Description of key information

EC50 (invertebrates) = 0.564 (C.I. 0.392 - 0.848) mg/L based on geometric mean measured concentrations, 48-hour, freshwater, OECD TG 202, 2017

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.564 mg/L

Additional information

Key study : OECD TG 202, 20178 : The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test and EU Method C.2 guidelines under GLP. The study was conducted in a closed system (sealed glass flasks) without headspace under semi-static conditions over a period of 48 hours with five concentration levels of the test item in the 6.25% to 100% saturated solution concentration range: prepared in a geometric series with a separation factor of 2. To reduce losses of the test item, the study was conducted under semi-static conditions in a closed system. A stock solution of 100% saturation was prepared with modified-Elendt M4 (according to OECD 202, Annex 3) with total hardness of 160 to 180 CaCO3/L with a nominal loading of 100 µL/L of the test item in duplicate one day prior to the start of the exposure and the renewal of the test solutions (at minus 24 and 0 hours) in tightly closed glass flasks with a lateral outlet. The glass flask (total volume ca. 1150 mL) were filled up to the top with approximately 1150 mL of the dilution water and closed with a screw cap. The test item (115 µL) was placed by pipette under the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 24 hours with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 2 hours for separation of undissolved test item. Thereafter, the saturated solution was removed by the lateral outlet of the flask (from the approximate bottom of the glass flask). Five test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25%, 50% and 100% of saturated solution. All test concentrations were visually clear throughout the exposure intervals. Twenty daphnids were exposed to each concentration level and the control. The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette. The concentrations of the test item were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all relevant concentration levels and the control. The measured concentrations of the test item were in the range of 64 to 74% of the 0 hour measured concentrations at 24 hours and 64 to 75% of the 24 hour fresh test solution measured concentrations at 48 hours. The geometric mean measured concentrations were calculated to be: 0.146, 0.242, 0.534, 1.05 and 2.00 mg/L. Since the measured test item concentrations did not remain within ± 20% of the nominal concentrations, the EC50-values are based upon the geometric mean measured concentrations. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled. The 48h-EC50 was 0.564 mg/L (C.I: 0.362 – 0.848 mg/L) based on geometric mean measured concentrations of the test item.