2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

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:

04 December to 19 March 2018

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Batch # AH03201
- Purity, including information on contaminants, isomers, etc.: 100%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 °C in the dark
- Expiry Date: 1 February 2019

Analytical monitoring:

yes

Details on sampling:

Samples were taken for quantitative analysis from the control and all test concentrations from the freshly prepared bulk test preparation at 0 and 24 hours and from the corresponding old or expired test preparations (replicates pooled) at 24 and 48 hours. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 20 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution of the test item. A series of dilutions was made from this saturated solution to give further test concentrations of 56, 32, 18 and 10% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0, 24 and 48 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

First instar daphnids from in-house laboratory cultures. Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 to 21°C

pH:

7.8 to 8.1

Dissolved oxygen:

8.7 to 9.1 mg/L

Salinity:

not applicable

Nominal and measured concentrations:

nominal concentrations of 10, 18, 32, 56 and 100% v/v, the resulting geometric mean measured concentrations for the four lower concentrations were 2.1, 3.6, 6.2 and 10 mg/L.

Details on test conditions:

In the definitive test 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.
Test Organism Observations
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

Range-finding Test
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in the tables below.
No immobilization was observed at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution, however, immobilization was observed at 100% v/v saturated solution.
A sub-lethal effect of exposure was observed at the test concentration of 10% v/v saturated solution at 24 hours. This response was trapping at surface.
Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 2.4 to 21 mg/L. A decline in measured test concentrations was observed at 48 hours to between 1.7 and 16 mg/L indicating that the test item was unstable under the conditions of the test. As such a semi-static testing regime was selected for the definitive test.

Verification of Test Concentrations
Chemical analysis of the freshly prepared 10, 18, 32 and 56% v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to range from 2.0 to 12 mg/L. Analysis of the corresponding old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 1.6 and 9.5 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 2.1, 3.6, 6.2 and 10 mg/L.
Measurable levels of test item were detected in the freshly prepared control preparation at 0 hours and in the corresponding old preparation at 24 hours. Given that the levels were significantly below the measured test concentration obtained at the lowest level employed, and that no adverse effects were observed in the control vessels throughout the test this was considered to have had no adverse effect on the outcome of the test.

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in the tables below.

Reported statistics and error estimates:

The EC50 value and associated confidence limits at 24 hours were calculated by Probit analysis using the binominal distribution. The LOEC and the NOEC at 24 hours were calculated using the Step-down Cochran-Armitage test procedure. The EC50 value and associated confidence limits at 48 hours were calculated by Weibull analysis using Linear Maximum-Likelihood regression. The LOEC and the NOEC at 48 hours were calculated using the Step-down Cochran-Armitage test procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Water Quality Measurements
Nominal Concentration (% v/v Saturated Solution)		0 Hours (Fresh Media)			24 Hours (Old Media)
		pH	mg O2/L	Temp °C	pH	mg O2/L	Temp °C
Control	R1	7.9	9.1	20	8	8.9	21
10	R1	7.9	9.1	20	8	9	20
18	R1	7.9	9.1	20	8	8.8	20
32	R1	7.9	9.1	20	8	8.8	20
56	R1	7.9	9	21	8	9	20
100	R1	8	9.1	21	8.1	9.1	20

Nominal Concentration (% v/v Saturated Solution)		24 Hours (Fresh Media)			48 Hours (Old Media)
		pH	mg O2/L	Temp °C	pH	mg O2/L	Temp °C
Control	R1	8.1	9.1	20	7.8	8.9	21
10	R1	8	9.1	20	8	8.9	21
18	R1	8	9.1	20	8	8.8	20
32	R1	8	9.2	21	8	8.9	20
56	R1	8	9	21	8	8.7	20
100	R1	8	8.9	21	8.1	8.8	20

 

Analytical Results for Test Samples
Time Point (Hours)	Nominal Concentration of Test Item in Test Sample (% v/v Saturated Solution)	Determined Concentration of Test Item in Test Sample (mg/L)
0	Control	0.582 / 1.04*
	10	2.71
	18	4.08
	32	6.62
	56	11.8
24 (old)	Control	0.0257 / 0.213*
	10	1.92
	18	3.37
	32	5.68
	56	9.51
24 (fresh)	Control	<LOQ
	10	2
	18	3.93
	32	6.86
	56	10.5
48 (old)	Control	<LOQ
	10	1.63
	18	3.17
	32	5.7
	56	9.02

LOQ = Limit of Quantification
*Duplicate sample stored frozen prior to analysis

 

 

Cumulative Immobilization Data and Observations in the Range-finding Test
Nominal Concentration (% v/v Saturated Solution)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	R1	R2	R1	R2	R1	R2	R1	R2
Control	0	0	5N	5N	0	0	5N	5N
0.10	0	0	5N	5N	0	0	5N	5N
1.0	0	0	5N	5N	0	0	5N	5N
10	0	0	4N 1T	5N	0	0	5N	5N
100	5	5	AI	AI	5	5	AI	AI

R = Replicate
N = Normal
T = Trapped at surface
AI = All daphnia immobilized

 

 

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	0	0	0	0	0	0	5 N	5 N	5 N	5 N
56	5	5	5	5	20	100	AI	AI	AI	AI
100	5	5	5	5	20	100	AI	AI	AI	AI

 

Nominal Concentration (% v/v Saturated Solution)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	1	3	1	5	25	5 N	4 N	2 N	4 N
32	2	1	0	1	4	20	3 N	4 N	5 N	4 N
56	5	5	5	5	20	100	AI	AI	AI	AI
100	5	5	5	5	20	100	AI	AI	AI	AI

R = Replicate
N = Normal
T = Trapped at surface
AI = All daphnia immobilized

Validity criteria fulfilled:

yes

Conclusions:

The 48-hr EC50 value for Daphnia magna has been reported as 6.5 mg/L based on geometric mean measure test concentrations

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.
Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.  A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 20 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.
Following a preliminary range-finding test and initial experiments, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of 20 °C to 21 °C under semi-static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the freshly prepared 10, 18, 32 and 56% v/v saturated solution test preparations at 0 and 24 hours showed measured test concentrations to range from 2.0 to 12 mg/L. Analysis of the corresponding old or expired test preparations at 24 and 48 hours showed measured test concentrations had declined, to between 1.6 and 9.5 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 2.1, 3.6, 6.2 and 10 mg/L.
Measurable levels of test item were detected in the freshly prepared control preparation at 0 hours and in the corresponding old preparation at 24 hours. Given that the levels were significantly below the measured test concentration obtained at the lowest level employed, and that no adverse effects were observed in the control vessels throughout the test this was considered to have had no adverse effect on the outcome of the test.

Analysis of the immobilization data by Probit analysis using the binominal distribution at 24 hours and by Weibull analysis using Linear Maximum-Likelihood regression at 48 hours the 48 -hr EC50 value was 6.5 mg/L based on the geometric mean measured test concentrations. The NOECs after 24 and 48 hours exposure were 6.2 and 2.1 mg/L respectively. The LOECS after 24 and 48 hours exposure were 10 and 3.6 mg/L respectively. The slope and standard error of the response curve at 48 hours were 7.9 (standard error = 0.028). Due to the nature of the data it was not possible to calculate the slope and error of response curve at 24 hours.

Description of key information

A daphnid acute toxicity test following OECD Guideline 202 reported a 48 -hour EC50 value of 6.5 mg/L based on geometric mean measured test concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

6.5 mg/L