(E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2014-12-16 to 2014-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 202 without any deviation.

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:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on June 03-05, 2013 / signed on November 05, 2013

Specific details on test material used for the study:

No additional information

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via GC-MS at the start (0 and 24 h) and at the end of both exposure intervals (24 and 48 h).
At the start of the exposure intervals (0 and 24 h), sampling was carried out after preparation of the test concentrations.
At the end of the exposure intervals (24 and 48 h), samples were taken directly from the test vessels.
The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Quality criteria for the analytical monitoring:
Recoveries of the test item should be within ± 20% of the nominal concentrations. The effect levels can be given based on the nominal concentrations, if this quality criteria is fulfilled.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution (10.0 mg/L of the test item were weighed out) was freshly prepared with ISO test water before the start of both exposure intervals (at 0 and 24 h).
The stock solution in the measuring flask was mixed thoroughly by inverting the measuring flask 100 times.
Application: 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 will be closed immediately with screw caps.
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), D-14195 Berlin, Germany.
- Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany.
- Age at study initiation: 2 to 24 h old daphnids from a healthy stock were used for the study. They were obtained by removing the juvenile daphnids from the culture vessels latest 22 h before the start of the exposure. The juveniles born within the 22 h preceding the exposure were used for the test after an acclimatisation phase of 2 hours in the dilution water. No first brood progeny was used for the test.
- Culture: 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 µE/m2/s
- Culture feeding: 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.
- Origin of the food algae: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany.
- Feeding during test: The daphnids were not fed during the study.

ACCLIMATION
- Acclimation period: At least 2 h in dilution water

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

255 to 260 mg/L as CaCo3

Test temperature:

19-20 °C

pH:

7.61-7.74

Dissolved oxygen:

9.03-9.17 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

- Nominal concentrations: 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L.
- Measured concentrations: See table 6.1.3/2 in "Any other information on results incl. tables".

Details on test conditions:

TEST METHOD:
A semi-static test design with a daily renewal of the test solutions in glass flasks sealed with screw caps (made from polypropylene) under diffuse light conditions was chosen to reduce losses of the test item concentrations by evaporation. The glass flasks were filled up with the test solutions having nearly no headspace.

TEST SYSTEM
- Test vessels/volume: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm, ca. 130 mL) with screw caps (made from polypropylene) were used. They were filled up with the test solutions having nearly no headspace to reduce contact with air and losses of the test item concentrations by evaporation.
- 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 (control and test material groups)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Renewal of the test solutions: The test solutions were renewed after 24 h. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by a pipette.

TEST MEDIUM / WATER PARAMETERS
- Dilution water: ISO Test water, according to OECD 202
- Culture medium: Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.
- Alkalinity: 260 mg CaCO3/L
- Conductivity: 639-648 µs/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/8 h dark cycle
- Light intensity: Diffuse light; light intensity of max. 20 µE/m2/s

EFFECT PARAMETERS MEASURED:
Biological parameters:
- Immobilisation and other observations: 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. Other relevant observations were not made.

Water Quality Parameters:
Dilution water: Prior to the start of the exposure (0 h) and the water renewal (24 h), the water quality parameters (i.e. pH value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured.
Test media: At the start of the exposure intervals (0 and 24 h), the water quality parameters of the fresh media (i.e. pH value, dissolved oxygen concentration) were measured in one additional replicate per concentration level and control. At the end of the exposure intervals (24 and 48 h), the water parameters of the old media were measured in all replicates per concentration level and control.
Temperature: The incubator temperature was recorded throughout the period of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Factor 2
- Range finding study: Test concentrations were selected based on the results of a non GLP preliminary range finding test. This test was conducted under semi-static conditions with three concentrations of the test item of 0.100, 1.00 and 10.0 mg/L and two replicates per concentration level with 10 daphnids each were tested in a closed system (glass flask completely filled up with the test solutions having no headspace). The percentage immobility was determined in all concentrations and control groups after 24 and 48 h of exposure.
- Results used to determine the conditions for the definitive study: After 24 h of exposure the total rate of immobilisation of daphnia was 100% in all three groups.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Cl: 1.99-2.81 mg/L

Details on results:

- Biological data:
Immobilization of daphnids for the definitive test was 0, 0, 35, 70 and 100 % (24 h); 0, 0, 55, 100 and 100 % (48 h) at 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L, respectively.
- Measured Exposure Concentrations during the Definitive Test:
The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 104 to 113 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 80 to 97 % of the nominal values. The measured test item concentrations were all within ± 20 % of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.
- Water Quality Parameters:
The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. 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: 2.05 mg/L (Cl 1.90 - 2.18 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h 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:

EC values and statistical analysis:
The EC100-values after 24 and 48 h were estimated directly from the observation data, without mathematical calculation.
The EC50-values after 24 and 48 h were calculated by sigmoidal dose-response regression. The 95 % confidence limits were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5. The concentration-effect relationships are shown graphically. The EC50 -value for the reference item and its confidence limits were calculated accordingly.

Table 6.1.3/1: Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

 

Nominal test item concentration   [mg/L]	IMMOBILISATION [%]
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
10.0	100	100	100	100	100	100	100	100	100	100
5.00	60	60	100	60	70	100	100	100	100	100
2.50	40	60	20	20	35	60	60	80	20	55
1.25	0	0	0	0	0	0	0	0	0	0
0.625	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

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

MV   = mean value

 

Table 6.1.3/2: Measured Concentrations of the Test Item during the Definitive Test

 

Sampling date	2014-12-16 0 hours Start of the exposure interval		2014-12-17 24 hours End of the exposure interval		2014-12-17 24 hours Start of the exposure interval		2014-12-18 48 hours End of the exposure interval
Start of analysis	2014-12-16		2014-12-17		2014-12-17		2014-12-18
Nominal test item concentration [mg/L]	Test item
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
10.0	11.3	113	8.53	85	11.3	113	Not determined*
5.00	5.18	104	4.37	87	5.26	105	4.79	96
2.50	2.66	106	2.43	97	2.65	106	2.41	96
1.25	1.33	106	1.07	86	1.41	113	1.12	90
0.625	0.662	106	0.501	80	0.698	112	0.561	90
Control	< SYSQL		< SYSQL		< SYSQL		< SYSQL

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

% = percent of the nominal concentration of the test item

SYSQL = System qualification limit (1 µg test item/L)

*Not determined due to 100% mortality after 24 h 

 

Validity criteria:

- In the control group, no daphnids were immobilized or showed any signs of disease or stress, e.g. discoloration or unusual behaviour such as trapping on the surface of the water, during the 48 h test period (required: not more than 10 per cent of the daphnids in the control).

- The dissolved O2 concentration at the end of the exposure intervals was ≥ 8.38 mg/L (required: ≥ 3 mg/L at the test end of the exposure intervals) in the test vessels of all tested concentration levels and the control.

Validity criteria fulfilled:

yes

Conclusions:

Based on analytically confirmed nominal concentrations of the test item, the 48 h-EC50 for Daphnia magna was 2.37 mg/L (95 % confidence limits: 1.99 - 2.81 mg/L).

Executive summary:

Study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the test substance to Daphnia magna, under semi-static conditions.

Test item was exposed to daphnids at the concentrations of 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L. for 48 hours. The no-treatment control daphnids were exposed to dilution water only. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. All test solutions were prepared in dilution water. The test temperature was 19-20 °C. The tested solutions were clear throughout the exposure period. Before definitive test, a 48-hour semi-static range-finding test was conducted and the percent immobilization was 100% at all three concentration levels (0.100, 1.00 and 10.0 mg/L).

 

The concentrations of the test item were analytically verified via GC-MS at the start of the exposure intervals (0 and 24 h) and at the end of the exposure intervals (24 and 48 h) in all concentration levels and the control. The measured concentrations of the test item at the start of the exposure intervals (0 and 24 h) were in the range of 104 to 113 % of the nominal values. At the end of the exposure intervals (24 and 48 h), the measured concentrations of the test item were in the range of 80 to 97 % of the nominal values. The measured test item concentrations were all within ± 20 % of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test. The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 h) and at the end of the exposure intervals (24 and 48 h), were within the acceptable limits. The validity criteria of the test guidelines were fulfilled.

 

Immobilization of daphnids for the definitive test was 0, 0, 35, 70 and 100 % (24 h); 0, 0, 55, 100 and 100 % (48 h) at 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L, respectively. 24 and 48 h EC50 values were 3.34 mg/L (CI: 2.50 - 4.41) and 2.37 mg/L (CI: 1.99 - 2.81). 24 and 48 h EC100 values were 10 and 5 mg/L, respectively. The EC50 -value of the reference item, potassium dichromate at 2.05 mg/L (CI 1.90 - 2.18 mg/L) after 24 h was within the prescribed concentration range of 0.6 - 2.1 mg/L.

 

Based on analytically confirmed nominal concentrations of the test item, the 48 h-EC50 for Daphnia magna was 2.37 mg/L (95 % confidence limits: 1.99 - 2.81 mg/L).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

From 2015-07-27 to 2015-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

QSAR value. The substance falls into the applicability domain of the QSAR model.

Justification for type of information:

1. SOFTWARE
iSafeRat® HA-QSAR

2. MODEL (incl. version number)
iSafeRat® holistic HA-QSAR v1.4

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC=CC(=O)C1C(C)=CCCC1(C)C
Experimental Log Kow value (slow-stirring method): 3.66 (25°C, pH 5.83)

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QMRF

6. ADEQUACY OF THE RESULT
See attached QPRF

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not applicable

Remarks:

(QSAR model)

Principles of method if other than guideline:

The acute toxicity to aquatic invertebrates was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 48-hour EC50 tests on daphnia, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 48-h EC50 for daphnia and is valid within the applicability domain defined in the QMRF.

GLP compliance:

no

Specific details on test material used for the study:

- Physical state: liquid
- Mode of action: Non-polar narcotic (MOA 1)
- Experimental Log Kow value (slow-stirring method): 3.66 (25°C, pH 5.83)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

Not applicable

Test organisms (species):

Daphnia sp.

Details on test organisms:

Not applicable

Test type:

other: QSAR

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Post exposure observation period:

Not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The temperatures varied from approximately 20 to 23 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the EC50 values found in experimental data.

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (>60% of the air-saturation value throughout the duration of the test). In exceptional cases where studies with oxygen concentrations lower than 60% were used, all aspects of the study were thoroughly evaluated in order to satisfy the evaluator that the effects found were not due to reduced oxygen concentration (i.e. the study would correctly receive a Klimisch score of 2 under the REACH Regulation [REACH, 2006]).

Salinity:

Not applicable.

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.

Details on test conditions:

Preferentially results from semi-static studies were used. However, substances tested using a static design were accepted (preferably accompanied by analytical measurements over the study period). For suspected volatile substances, only tests performed in closed vessels were accepted unless accompanying analytical monitoring proved such a design was not necessary.

Reference substance (positive control):

no

Remarks:

QSAR model

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 5.0-6.1 mg/L

Details on results:

The predicted value is reliable since the test substance falls within the applicability domain of the model. The water solubility value of the test substance is within descriptor domain of the model between log water solubility (in log (mol/L)) of -4.70 to 0.87. Moreover the test substance can be well predicted by non-polar narcotic compounds (MOA 1) HA-QSAR model.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

95% CL: 5.0-6.1 mg/L
QSAR statistical parameters are given in the QMRF and the QPRF.

No additional information

Validity criteria fulfilled:

yes

Remarks:

The substance falls into the applicability domains of the QSAR model.

Conclusions:

The 48-h EC50 based on mobility was determined to be 5.5 mg/L with 95%-Confidence Limit between 5.0 and 6.1 mg/L.

Executive summary:

A QSAR prediction was performed to assess the acute toxicity of the test substance to daphnid. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The criterion predicted was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

 

The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). Use of this MOA for substituted α,β-unsaturated ketones has previously been demonstrated as acceptable in a position paper (KREATiS, 2015; in "Attached background material"). The QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour tests on daphnids, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The water solubility of the substance given as input was derived from the experimental log Kow value, measured at 3.66 with a slow-stirring method.

The substance falls within the applicability domain of the model as demonstrated in the QPRF. The 48-h EC50 based on mobility was determined to be 5.5 mg/L with 95%-Confidence Limit between 5.0 and 6.1 mg/L.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 2.37 mg/L (95% CL: 1.99 - 2.81 mg/L) based on analytically confirmed nominal concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.37 mg/L

Additional information

To assess the short-term toxicity of the registered substance to aquatic invertebrates, two data are available.

The first study (NOACK, 2015) was assessed as the key study and was performed on the registered substance according to OECD Guideline 202 with GLP statement. In this study, the test substance was exposed to Daphnia magna at the concentrations of 0 (control with dilution water only), 0.625, 1.25, 2.50, 5.00 and 10.0 mg/L for 48 hours, under semi-static conditions (four replicates per treatment; five daphnids per replicate). According to the results of this study, the 48h-EC50 was determined at 2.37 mg/L (95% CL: 1.99 - 2.81 mg/L), based on analytically confirmed nominal concentrations.

The second data (KREATiS, 2015), assessed as a supporting data, is a QSAR. This QSAR prediction (iSafeRat holistic approach v1.4) was performed on the registered substance, to assess the acute toxicity of the substance to daphnids. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modelling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following OECD Guideline 202. The immobility of the daphnids was determined using a validated QSAR for the Mode of Action in question, (MOA 1, non-polar narcosis). Use of this MOA for substituted α,β-unsaturated ketones has previously been demonstrated as acceptable in a position paper (KREATiS, 2015; available in the Endpoint Study Record). This QSAR is based on validated data for a training set of 58 chemicals derived from 48-hour EC50 tests on daphnids, for which the concentrations of the test substance had been determined by chemical analyses over the test period. The water solubility of the substance given as input was derived from the experimental log Kow value, measured at 3.66 with a slow-stirring method. The substance falls within the applicability domain of the model. The 48h-EC50 based on mobility was determined to be 5.5 mg/L (95% CL: 5.0 - 6.1 mg/L). This QSAR result supports the key study performed on the same substance with an EC50 value in the same order of magnitude, considering the biological and experimental variabilities (factor 2 - 3) . Therefore, this QSAR is considered relevant and sufficient to assess, alone, the toxicity of the registered substance to aquatic algae, without the use of an experimental study.

In conclusion, according to the key experimental study (NOACK, 2015), the 48h-EC50 value on Daphnia magna is 2.37 mg/L (95% CL: 1.99 - 2.81 mg/L).