Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene

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:

The study was conducted between 03 April 2018 and 20 April 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 (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 14-0383
Physical state/Appearance: Colorless liquid
Storage Conditions: Stored cold at ~ 4 °C in darkness

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were analyzed immediately. A set of duplicate samples was taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

Range-finding Test
A nominal amount of test item (550 mg) was dispersed in 11 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 Gelman Acrocap filter (first approximate 500 mL 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 further test concentrations of 0.10, 1.0 and 10% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (550 mg) was dispersed in 11 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 Gelman Acrocap filter (first approximate 500 mL discarded in order to pre condition the filter) to give a 100% v/v saturated solution. The 100% v/v saturated solution was diluted with test media to produce a 10% v/v saturated solution. A series of dilutions of the 10% v/v saturated solution produced further test concentrations of 1.0, 1.8, 3.2 and 5.6% v/v saturated solution.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 2) in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

18 to 22 °C with a maximum deviation of ±1 °C

pH:

7.7-7.8

Dissolved oxygen:

7.7-7.8 mg O2/L

Nominal and measured concentrations:

Range-finding Test:
Nominal concentration: 0.10, 1.0, 10 and 100% v/v saturated solution

Definitive Test:
Nominal concentration: 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.089 to 0.96 mg/L.

Details on test conditions:

Reconstituted Water – Elendt M7 Medium
Ingredient Final Concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075

The pH of the prepared media was 7.9 ±0.3 and stored at approximately 21 ºC

Preliminary Media Preparation Trial
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.

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 9.5 mg/L could be obtained using a saturated solution method of preparation.
The test concentrations to be used in the definitive test were determined by a preliminary range finding test.
In the range finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
In the range finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 150 mL of test media and was sealed with a glass stopper leaving no headspace due to the potentially volatile nature of the test item. After 24 and 48 hours the number of immobilized daphnids was recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for immediate chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions.

Definitive Test
Based on the results of the range finding test the following test concentrations were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution.
The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 48 hours.

Exposure Conditions
As in the range finding test 150 mL stoppered glass conical flasks containing approximately 150 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 sealed with a glass stopper and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and 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.
The test preparations were not renewed during the exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.57 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 0.91-11.46 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.63 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed at the test concentrations of 0.10 and 1.0% v/v saturated solution, however, immobilization was observed at 10 and 100% v/v saturated solution.
A sub lethal effect of exposure was observed in the 10% v/v saturated solution test concentration at 24 hours. The response was reduced mobility.
Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.02 to 12.7 mg/L. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.089 to 0.96 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on 0 hour measured test concentrations.

Immobilization Data
Analysis of the immobilization data by Probit analysis using Linear Maximum Likelihood regression at 24 and 48 hours based on the 0 Hour measured test concentrations gave the following results:
Time (hour) EC50 (mg/L) 95% Confidence limits(mg/L)
24 1.57 0.91 – 11.46
48 0.63 Not determined due to the nature of the data
The NOEC after 24 and 48 hours exposure was 0.16 mg/L. The LOEC after 24 and 48 hours exposure was 0.29 mg/L.
The slopes and their standard errors of the response curves at 24 and 48 hours were 2.12 (standard error = 0.18) and 3.00 (standard error = 0.062) respectively.

Sub-Lethal Effects
A sub lethal effect of exposure was observed at 0.49 and 0.96 mg/L. This response was reduced mobility.

Validation Criteria
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria
The results of the water quality measurements are given in Table 3.
Temperature was maintained at 21 °C to 23 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 603 to 793 Lux.

Observations on Test Item Solubility
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control (Envigo study number LK67NP) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 48 hours and the Trimmed Spearman-Karber method at 24 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
Time Point
(Hours) EC50(mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC(mg/L)
24 0.79 0.73 - 0.86 0.56 1.0
48 0.75 0.56 - 1.0 0.56 1.0

The NOEC is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item*.

NOEC = No observed effect concentration
LOEC = Lowest observed effect concentration
*The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24 Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L

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
	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2
Control	0	0	5N	5 N	0	0	5 N	5 N
0.10	0	0	5 N	5 N	0	0	5 N	5 N
1.0	0	0	5 N	5 N	0	0	5 N	5 N
10	2	4	3 N	1 R	5	5	AI	AI
100	5	5	AI	AI	5	5	AI	AI

Rep  = Replicate

N      = Normal

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)	0-Hour Measured Concentration (mg/L)	24 Hours
		Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)									Observations
		Rep1		Rep2	Rep3	Rep4	Total	%			Rep1		Rep2		Rep3	Rep4
Control	-	0		0	0	0	0	0			5N		5 N		5 N	5 N
1.0	0.089	0		0	0	0	0	0			5 N		5 N		5 N	5 N
1.8	0.16	0		0	0	0	0	0			5 N		5 N		5 N	5 N
3.2	0.29	1		0	1	0	2	10			4 N		5 N		4 N	5 N
5.6	0.49	0		0	1	2	3	15			5 N		5 N		4 N	3 R
10	0.96	3		2	1	0	6	30			2 R		3 N		4 N	5 N
Nominal Concentration (% v/v Saturated Solution)	0-Hour Measured Concentration (mg/L)	48 Hours
		Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)								Observations
		Rep1		Rep2	Rep3	Rep4	Total		%	Rep1		Rep2		Rep3		Rep4
Control	-	0		0	0	0	0		0	5 N		5 N		5 N		5 N
1.0	0.089	0		0	0	0	0		0	5 N		5 N		5 N		5 N
1.8	0.16	0		0	0	0	0		0	5 N		5 N		5 N		5 N
3.2	0.29	1		0	1	0	2		10	4 N		5 N		4 N		5 N
5.6	0.49	5		2	1	5	13		65	AI		3 N		4 N		AI
10	0.96	3		2	4	2	11		55	2R		2 N 1R		1 N		3 N

Rep    =Replicate

N          = Normal

R     = Reduced Mobility

AI     = All daphnia immobilized

Water Quality Measurements

Nominal Concentration (% v/v Saturated Solution)	0-Hour Measured Concentration (mg/L)		0 Hours			24 Hours	48 Hours
			pH	mg O2/L	Temperature °C	Temperature ºC	pH	mg O2/L	Temperature °C
Control	-	Rep1	7.7	9.1	21	23	7.9	8.8	21
1.0	0.089	Rep1	7.7	9.1	21	22	7.8	8.6	21
1.8	0.16	Rep1	7.8	9.2	21	22	7.7	8.1	21
3.2	0.29	Rep1	7.8	9.1	21	22	7.6	6.7*	21
5.6	0.49	Rep1	7.8	9.1	21	22	7.6	7.8	21
10	0.96	Rep1	7.8	9.0	21	22	7.7	8.0	21

Rep = Replicate

*Low value considered to be due to the fact that the test vessels were sealed, no impact on test

Validity criteria fulfilled:

yes

Conclusions:

The 48 h EC50 (immobilisation) value with Daphnia magna is 0.63 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of FRET 14-0383 to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. 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 9.5 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, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution for 48 hours at a temperature of 21 °C to 23 °C under static test conditions. The test item solutions were prepared by stirring an excess (50 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 Gelman Acrocap filter, (first approximate 500 mL 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 required test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.089 to 0.96 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on the 0‑Hour measured test concentrations.

Exposure of Daphnia magna to the test item gave the following results based on the 0‑Hour measured test concentrations:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	0.63	Not determined due to the nature of the data	0.16	0.29

Description of key information

A study was performed to assess the acute toxicity of FRET 14-0383 to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. 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 9.5 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, 20 daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 1.8 , 3.2, 5.6 and 10% v/v saturated solution for 48 hours at a temperature of 21 °C to 23 °C under static test conditions. The test item solutions were prepared by stirring an excess (50 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 Gelman Acrocap filter, (first approximate 500 mL 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 required test concentrations. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.089 to 0.96 mg/L. There was no significant change in the measured concentrations at 48 hours and so the results are based on the 0‑Hour measured test concentrations.

Exposure of Daphnia magna to the test item gave the following results based on the 0‑Hour measured test concentrations:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	0.63	Not determined due to the nature of the data	0.16	0.29

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.63 mg/L