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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017 to 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity 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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

- Treatment of test material prior to testing (e.g. warming, grinding): A nominal amount of test item (2200 mg) was dispersed in 22 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and 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. A series of dilutions was made from this saturated solution to give test solutions of 3.13, 6.25, 12.5, 25 and 50% v/v saturated solution.
Each test solution was mixed with a flat bladed stirrer for one minute 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, 72 and 96 hours.

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Water samples were taken from the control and each test group, from fresh and old media throughout the 96-Hour test period, for quantitative analysis. The samples at 0 and 72 hours (fresh media) and 24 hours (old media) were stored frozen prior to analysis with the 96-Hour samples. All other samples were stored frozen prior to analysis. Duplicate samples at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Fish were exposed, in groups of seven, to an aqueous solution of the test item at nominal concentrations of 3.13, 6.25, 12.5, 25 and 50% v/v saturated solution for a period of 96 hours at a temperature of 14 to 15 C under semi static test conditions. The test item solution was 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 (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre condition the filter) to produce a 100% v/v saturated solution of the test item. The number of mortalities and any sub lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 09 March 2018. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 02 April 2018 to 09 April 2018. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at 14 °C to 15 °C with a dissolved oxygen content of greater than or equal to 9.7 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 25 hours prior to the start of the definitive test. There was no mortality in the seven days prior to the start of the test and the fish had a mean standard length of 5.5 cm (standard deviation = 0.3) and a mean body weight of 1.31 g (standard deviation = 0.24) at the end of the definitive test. Based on the mean body weight value this gave a loading rate of 0.46 g body weight/liter (volume in 24 hours).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

approximately 140 mg/L as CaCO3

Test temperature:

14-15C

pH:

7.4-7.9

Dissolved oxygen:

8.9-10.1 mg/L

Nominal and measured concentrations:

Nominal: 3.13, 6.25, 12.5, 25, 50 %v/v

Geometric Mean Measured: 0.53, 1.1, 2.3, 4.4, and 9.7 mg/L

Details on test conditions:

The test water used for the initial epxeriment and definitive test was the same as that used to maintain the stock fish. Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

For each control and test concentration, 25 to 30 liter glass exposure vessels containing 20 liters of test media were used. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 14 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The control group was maintained under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.

For each control and test concentration, 25 to 30 liter glass exposure vessels containing 20 liters of test media were used. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 14 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure. The control group was maintained under identical conditions but not exposed to the test item.

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

2.9 mg/L

95% CI:

2.4 - 3.5

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.9 mg/L

95% CI:

2.4 - 3.5

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Details on results:

Analysis of the freshly prepared test media at 0 and 72 hours showed measured test concentrations to range from 0.71 to 12 mg/L. A decline in measured test concentration was observed in the old or expired test media at 24 and 96 hours to between 0.38 and 7.6 mg/L and hence it was considered appropriate to calculate the results based on the time-weighted mean measured test concentration only in order to give a “worst case” analysis of the data.

Reported statistics and error estimates:

Analysis of the mortality data by Trimmed Spearman-Karber method at 72 and 96 hours based on the geometric mean measured test concentrations.

Sublethal observations / clinical signs:

Cumulative Mortality Data in the Definitive Test
Geometric Mean Measured Test Concentration (mg/L)	Cumulative Mortality (Initial Population = 7) (Hours)							Mortality (%) @ 96 Hours
	1	3	6	24	48	72	96
Control	0	0	0	0	0	0	0	0
0.53	0	0	0	0	0	0	0	0
1.1	0	0	0	0	0	0	0	0
2.3	0	0	0	0	0	1	1	14
4.4	0	0	0	4	7	AD	AD	100
9.7	0	0	0	7	AD	AD	AD	100

AD = All Dead

Sub-lethal Effects of Exposure in the Definitive Test
Geometric Mean Measured Test Concentration (mg/L)	Sub-lethal Effects	Time (Hours)
		1	3	6	24	48	72	96
Control	No abnormalities detected	7	7	7	7	7	7	7
0.53	No abnormalities detected	7	7	7	7	7	7	7
1.1	No abnormalities detected	7	7	7	7	7	7	7
2.3	No abnormalities detected	7	7	7	7	4	0	0
	Increased pigmentation	0	0	0	0	3	6	0
	Increased pigmentation and swimming at bottom	0	0	0	0	0	0	5
	Increased pigmentation and loss of equilibrium	0	0	0	0	0	0	1
4.4	No abnormalities detected	7	7	7	0	0	AD	AD
	Increased pigmentation and swimming at bottom	0	0	0	3	0	AD	AD
	Moribund	0	0	0	2	2	AD	AD
9.7	Increased pigmentation and swimming at bottom	7	7	7	AD	AD	AD	AD

AD = All Dead

Water Quality Measurements
Geometric Mean Measured Test Concentration (mg/L)	Time (Hours)
	0 Hours (Fresh Media)			24 Hours (Old Media)
	pH	mg O2/L	Temp °C	pH	mg O2/L	Temp °C
Control	7.4	11.5	14	7.7	8.9	14
0.53	7.5	11.5	14	7.8	9.7	14
1.1	7.5	11.4	14	7.8	9.7	14
2.3	7.5	11.4	14	7.9	9.5	14
4.4	7.6	11.2	14	7.8	9.5	14
9.7	7.6	10.9	15	7.9	9.9	14

Geometric Mean Measured Test Concentration (mg/L)	Time (Hours)
	24 Hours (Fresh Media)			48 Hours (Old Media)
	pH	mg O2/L	Temp °C	pH	mg O2/L	Temp °C
Control	7.5	11.1	14	7.4	9.2	14
0.53	7.5	11.3	14	7.5	9.8	14
1.1	7.5	11.3	14	7.6	9.8	14
2.3	7.6	11.3	14	7.6	9.7	14
4.4	7.6	11.2	14	7.7	10.1	14
9.7	AD	AD	AD	AD	AD	AD

Geometric Mean Measured Test Concentration (mg/L)	Time (Hours)
	48 Hours (Fresh Media)			72 Hours (Old Media)
	pH	mg O2/L	Temp °C	pH	mg O2/L	Temp °C
Control	7.7	11.4	14	7.7	9.2	14
0.53	7.7	11.3	14	7.7	9.4	14
1.1	7.7	11.3	14	7.8	9.8	14
2.3	7.7	11.3	14	7.8	9.7	14
4.4	AD	AD	AD	AD	AD	AD
9.7	AD	AD	AD	AD	AD	AD

Geometric Mean Measured Test Concentration (mg/L)	Time (Hours)
	72 Hours (Fresh Media)			96 Hours (Old Media)
	pH	mg O2/L	Temp °C	pH	mg O2/L	Temp °C
Control	7.6	11.2	14	7.5	9.1	14
0.53	7.6	11.2	14	7.6	9.5	14
1.1	7.6	11.2	14	7.7	9.7	14
2.3	7.6	11.2	14	7.7	9.7	14
4.4	AD	AD	AD	AD	AD	AD
9.7	AD	AD	AD	AD	AD	AD

AD = All Dead

 

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	<LOQ
	3.13	0.706
	6.25	1.5
	12.5	3.06
	25	5.6
	50	12.3
24 (old)	Control	<LOQ
	3.13	0.376
	6.25	0.729
	12.5	1.49
	25	3.52
	50	7.57
72 (fresh)	Control	<LOQ
	3.13	0.716
	6.25	1.54
	12.5	3.06
96 (old)	Control	<LOQ
	3.13	0.412
	6.25	0.799
	12.5	1.97

Validity criteria fulfilled:

yes

Remarks:

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV (6.1 mg O2/L) in the control and test vessels.

Conclusions:

The 96-hr LC50 value for rainbow trout was determined to be 2.9 mg/L based on geometric mean measured test concentrations.

Executive summary:

An OECD 203 study was conducted with MCS-1562 exposed to the rainbow trout (Oncorhynchus mykiss). The 96 -hr LC50 value was determined to be 2.9 mg/L as was based on geometric mean measured concentrations.

Description of key information

An OECD 203 study was conducted with MCS-1562 exposed to the rainbow trout (Oncorhynchus mykiss). The 96 -hr LC50 value was determined to be 2.9 mg/L as was based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

2.9 mg/L