Reaction mass of rel-{(1R,2S)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol and rel-{(1S,2R)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2013 - July 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

A semi-static study set-up was adopted with renewal of exposure solutions every 24 hour period.

Fresh and Aged media were sampled and analysed for all test concentrations and Control from two intervals : 0 hour (fresh media) / 24 hour (aged media) and 72 hour (fresh media) / 96 hour (aged media).

Vehicle:

no

Details on test solutions:

At the start of the test and first media renewal, ca 1000 mg of the test substance was
added to 5000 mL of treated mains water, slow stirred for ca 24 hour and then
filtered through a 0.45 μm filter, discarding the initial 100 mL of filtrate, to give the
100% saturated solution test concentration. Dilutions were prepared from the 100%
saturated solution to give the 0.625, 1.25, 2.5, 5.0 and 10% saturated solution test
concentrations.

At each remaining media renewal, the test concentrations were prepared in a similar manner with the exception that the 100% saturated solution was prepared by stirring ca 500 mg of test substance in 2500 mL of treated mains water.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Healthy Oncorhynchus mykiss (rainbow trout) were supplied by Brow Well Fisheries, United Kingdom, details of which are maintained in Smithers Viscient (ESG) Ltd. records.

The fish were acclimatised to the test conditions for at least 12 days prior to testing.
The mortality rate of the stock batch of fish was 0% in the seven days prior to the definitive test.
Fish were selected at random from the holding tank and allocated to the test vessels at the start of each phase of testing. The fish were not fed for at least 24 hours before the start of the definitive test and were not fed throughout the test.
The total length and wet weight of ten randomly selected fish from the stock fish were determined periodically to ensure fish were of a suitable size and weight for testing and to ensure the correct testing volumes were used so as not to exceed the recommended loading rate.
At the end of the test, the total length and wet weight of ten fish, selected at random from those used in the test, were determined.

The Oncorhynchus mykiss were received at the test facility on 30 December 2013.
The mean total length and mean wet weight of a sub-sample of the remaining fish
used in the test at the end of the test is presented below.
Mean total length:
Minimum - maximum: 5.8 cm 5.2 – 6.2 cm
Mean wet weight:
Minimum - maximum: 1.89 g 1.05 – 2.21 g
Fish loading rate: 0.53 g/L
The fish were considered acceptable for use on the study as the fish weight to volume
ratio was <1.0 g/L

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Total hardness of water was controlled at each of the renewal periods :

Values expressed as CaCO3 mg/L :
0 hour 95
24 hour 110
48 hour 105
72 hour 116

Test temperature:

Range measured throughout the 96 hour exposure period : 13.6 - 14.6 °C

pH:

pH range in :
Control : pH 7.62 - 7.91
0.31 mg/L : pH 7.63 - 7.95
0.65 mg/L : pH 7.64 - 7.95
1.2 mg/L : pH 7.60 - 7.99
2.5 mg/L : pH 7.60 - 8.01
4.2 mg/L : pH7.71 - 8.02

Dissolved oxygen:

Dissolved Oxygen Range in mg O2/L :
Control : 8.80 - 10.26
0.31 mg/L : 8.70 - 10.25
0.65 mg/L : 8.90 - 10.30
1.2 mg/L : 8.55 - 10.25
2.5 mg/L : 8.95 - 10.35
4.2 mg/L : 9.72 - 10.25

Details on test conditions:

Definitive Test
Based on the results of the range-finding test, a definitive test was conducted at nominal test substance concentrations of 0.625, 1.25, 2.5, 5.0 and 10% saturated solution. A control group was also included.
The test was conducted in a temperature controlled laboratory (15 ± 2ºC) with a 16 hour light:8 hours dark lighting cycle. The test vessels were aerated to ensure oxygen concentration was maintained.
Single 30 L constructed glass aquaria, each fitted with a clear plastic lid, containing 25 litres of media, were used for the control and test vessels.
The test was conducted using semi-static test conditions with daily renewal of test media during the test.
At the start of the test and first media renewal, ca 1000 mg of the test substance was added to 5000 mL of treated mains water, slow stirred for ca 24 hour and then filtered through a 0.45 µm filter, discarding the initial 100 mL of filtrate, to give the 100% saturated solution test concentration. Dilutions were prepared from the 100% saturated solution to give the 0.625, 1.25, 2.5, 5.0 and 10% saturated solution test concentrations (see Table overleaf). At each remaining media renewal, concentrations were prepared in a similar manner with the exception that the 100% saturated solution was prepared by stirring ca 500 mg of test substance in 2500 mL of treated mains water. The volume prepared was reduced due to 100% mortality at the 10% saturated solution test concentration.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CI : 2.7 - 3.9 mg/L

Details on results:

The 96-hour LC50 value was calculated to be 3.2 mg/L, based on geometric mean measured test concentrations. The NOEC was considered to be 0.31 mg/L.

Sublethal observations / clinical signs:

The LC50 values and the corresponding NOEC values are presented in the following table. Values are presented in terms of geometric mean measured concentrations.

Toxicity value (mg/L)
Parameter	24 hours	48 hours	72 hours	96 hours
LC50	3.2 (2.7-3.9)	3.2 (2.7-3.9)	3.2 (2.7-3.9)	3.2 (2.7-3.9)
NOEC	1.2	1.2	0.31	0.31

Based on geometric mean measured concentrations, the 96-hour LC50 was determined to be 3.2 mg/L. The NOEC was considered to be 0.31 mg/L. The lowest concentration at which 100% mortality occurred was 4.2 mg/L. The lowest concentration at which there were significant sub-lethal effects was 0.65 mg/L. Sub-lethal effects of exposure were observed at test concentrations of 0.65 mg/L and above. A single fish was observed with increased pigmentation (a mild effect) at 0.65 mg/L. Mild symptoms of increased pigmentation and lethargy were observed at 1.2 mg/L. At the 2.5 mg/L test concentration, mild symptoms of increased pigmentation, swimming nose up and lethargy were observed although severe symptoms of barrel/disorientated swimming were also observed. Moribund fish were also observed at 4.2 mg/L.

The validity criteria for control mortality (less than 10%, actual = 0%) and dissolved oxygen (>60 % ASV, actual >88%) were both satisfied. The test was therefore considered to be valid.

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 value was calculated to be 3.2 mg/L, based on geometric mean measured test concentrations. The NOEC was considered to be 0.31 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 2014 - April 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Chemicals-Fish acute toxicity test” (GB/T 27861-2011); Chemicals- Rare minnow (Gobiocypris rarus) acute toxicity test (GB/T 29763-2013)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

The test was conducted under static conditions.

The Control, and nominal exposure concentrations of 10.0, 20.0, 40.0, 60.0, 80.0 and 100 mg/L were sampled for analysis at Time 0, 24, 48, 72 and 96 hours.

On each occasion, one sample was analysed after pre-treatment; the remaining samples were retained in case further analysis would be required.

Pre-treatment method of the sample :
50.0 ml collected water samples were extracted twice with 25.0 mL hexane. The combined upper organic phases were diluted to 50.0 mL by hexane, and then filtrated by 0.22 μm millipore membrane (discarding the initial 10.0 mL filtrates). The final solution were analysed by GC-FID.

Vehicle:

no

Details on test solutions:

Preparation of the Test Solutions :
In the range-finding test, the test solution of GR-50-1408 was prepared by directly adding appropriate amounts of GR-50-1408 in dilution water and then facilitating its dispersion by strring for 24 h.

In the definitive test, the solutions were prepared by adding appropriate amount of the test substance to the test medium in glass aspirator bottles with appropriate size. The mixing aspirator bottle was stoppered and the aqueous test substance mixture was stirred for 24 hrs on a magnetic stir-plate and a teflon stir-bar at room temperature (23 ºC - 25ºC) in the dark. The vortex height was set at approximately 10% of the static liquid height. At the end of the 24 h, stirring was stopped and the test substance was allowed to settle down. The mixture was allowed to settle for 1 hour at room temperature. Then the aqueous portion removed through the outlet of the aspirator stirring bottle without further filtration to remove the remaining undissolved test substance. The test solution was colourless and clear.

According to the purity of the test substance (87.8%, provided by sponsor), the test substance was converted to 100% active ingredient into dilution water.

Following a Range-Finder study, the following nominal test concentrations were chosen for the definitive study :
Control (no test item)
10.0 mg/L
20.0 mg/L
40.0 mg/L
60.0 mg/L
80.0 mg/L
100 mg/L

Dilution Medium :
Good quality tap water which had been dechlorinated for at least 24 hours was used. The total hardness of the test water was 145 mg (CaCO3)/L, oxygen concentration was about 98% of the air saturation value and pH was 7.82 at room temperature. Characteristics of the dilution water are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control.

Test organisms (species):

other: Rare minnow - Gobiocypris Rarus

Details on test organisms:

The test species of Rare minnow, Gobiocypris rarus (Batch No.: F20141215), were obtained as fry from a fish supplier of Institute of Hydrobiology, Chinese Academy of Sciences.
Feeding fry were at least held for 12 days in holding tanks supplied with a continuous flow of aerated water before being used for testing. Fish to be used for testing were held for 7 days in water of the quality and temperature to be used in the test.
The fish were fed daily during the holding period on proprietary fish food. They were held without food for approximately 24 hours before being placed into the test vessels.
During the holding period the tanks were inspected daily and any debris or unhealthy or dead fish removed.
After the 48 hour settling-in period, no mortality was observed following 7 days. So the batch of fish was accepted.
The average wet-weight and length of the fish used for the test was 0.287g and 2.68cm.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

During the whole test period, the total hardness of the control mediums and test mediums was in range of 133 to 147 mg (CaCO3)/L.

Test temperature:

During the test, the temperature of the test mediums was maintained in the range of 23.1°C to 23.5°C.

pH:

During the whole test period, the pH values of the control mediums and test mediums were between 7.90 and 8.36.

Dissolved oxygen:

During the whole test period, the dissolved oxygen values of the control mediums and test mediums varied from 70% - 95% of the air saturation at the test temperature.

Nominal and measured concentrations:

The study was performed under static conditions.

The mean exposure concentrations determined over the 96 hour test period were, as follows :

Nominal Exposure Concn. Mean Concn. Detected (% of Nominal)
Control Not Detected
10.0 mg/L 8.78 mg/L (87.8%)
20.0 mg/L 16.8 mg/L (84.0%)
40.0 mg/L 34.4 mg/L (86.0%)
60.0 mg/L 51.2 mg/L (85.3%)
80.0 mg/L 60.5 mg/L (75.6%)
100 mg/L 65.7 mg/L (65.7%)

Details on test conditions:

Range-Finding Test :
The range-finding test, carried out under static conditions, was conducted to determine the range of concentrations for the subsequent definitive test.

In the range-finding test, groups of fish (5 per group) were exposed to the test solutions with nominal concentration of 0.10, 1.0, 10.0 and 100mg/L. One control group was also included in the study using test water without the test substance. For each test tank 3 L test solution was filled. No replicates were used.

The test fish were randomly chosen and put in appropriate test solutions after the temperature has been adjusted to the required value. This was done in 30 minutes.
During the test, the following conditions were maintained:
- Light: 16 hours photoperiod daily (light intensity: 1000 to 1500 lux)
- Temp: 23.1-23.5°
- Oxygen conc: 80%~95% of the air saturation; No aeration
- Feeding: none

Test duration was 96 hours. Dead fish were removed at least once daily and discarded. The mortalities of the fish were recorded at 24, 48, 72 and 96 h, and then the maximum concentration causing no mortality (96h-LC0) and the minimum concentration causing 100% mortality (LC100) were determined.

Definitive Test :
The test substance was found to be stable in water during 96h therefore static method was adopted in the definitive test. Based on the results of the range-finding test, the concentrations of 10.0, 20.0, 40.0, 60.0, 80.0 and 100 mg/L were assigned in the definitive test. Synchronously blank control was used. No replicate was assigned for each treatment group and control group, while the initial number of testing fish was 10 for each group.

The test fish were randomly chosen and put in different test solutions after the temperature had been adjusted to the required value. This was done in 30 minutes.

During the test, the following conditions were maintained:
- Light: 16 hours photoperiod daily (light intensity: 1000 to 1500 lux)
- Temp: 23.1-23.5°
- Oxygen conc: 70%~95% of the air saturation; No aeration
- Feeding: none

A 3, 6, 24, 48, 72 and 96 h, the mortalities of the fish were recorded and observations on the individual behaviour was performed. Meanwhile, measurements of pH, dissolved oxygen and temperature were carried out and recorded daily.

Fish loading :
0.9-1.0 g fish (wet weight) per litre of test medium met the test validity criteria of maximum loading of 1.0 g fish/litre for static and semi-static test.

Reference substance (positive control):

yes

Remarks:

Potassium DiChromate

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

8.78 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

34.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

18.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI : 14.8 - 22.6 mg/L

Details on results:

During the whole test period, the pH values of the control mediums and test mediums were between 7.90 and 8.36, and the Dissolved Oxygen (DO) values varied from 70% - 95% of the air saturation at the test temperature, and the total hardness was in the range of 133 to 147 mg (CaCO3)/L. During the test, the temperature of the test mediums was maintained in the range of 23.1 ºC to 23.5 °C.

During the whole test period, all fish in the control and treatment of 10.0 mg/L were alive and appeared normal. Effects (Fish lying on side or back) occurred at nominal test concentrations at 20.0 mg/L. All fish at the 40.0 mg/L (nominal) exposure concentration, and above, were dead by 24 hours of exposure.

Results with reference substance (positive control):

K2Cr2O7 was used as the test substance and the resulting 24 h-LC50 was 269 mg/L.
(Acceoted range : 24 h-LC50 of K2Cr2O7 in the range of 200 to 400 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

The results showed that under valid static test conditions, the 96 h-LC50 of the test substance to fish (rare minnow, Gobiocypris rarus) is 18.3 mg/l with 95% confidence limit of 14.8 mg/l - 22.6 mg/l (based on measured concentration). The maximum tested concentration causing no mortality (96 h-LC0) is 10.0 mg/l (measured concentration 8.78 mg/l). The minimum concentration causing 100% mortality (96h-LC100) is 40.0 mg/l (measured concentration 34.4 mg/l) i.e:
96 h-LC50 = 18.3 mg/l (based on measured concentration)
96 h-LC0 = 10.0 mg/l (measured concentration 8.78 mg/l)
96 h-LC100 = 40.0 mg/l (based on measured concentration 34.4 mg/l)

Description of key information

 

In order to determine the 96-hour acute toxicity of the registration substance to fish, a 96-hour acute toxicity of GR-50-1408 to the freshwater fish species Oncorhynchus mykiss (rainbow trout) was determined under semi-static test conditions in accordance with the requirements of OECD Chemicals Testing Guideline No. 203 Fish, Acute Toxicity Test (adopted 17 July 1992). The 96-hour LC50 value was calculated to be 3.2 mg/L (95% CI : 2.7 - 3.9 mg/L), based on geometric mean measured test concentrations. The NOEC was considered to be 0.31 mg/L.

 

A supporting study has also been reported. This test was designed to determine the acute toxicity of the registration substance to fish (Chinese Rare Minnow, Gobiocyrpris rarus). The test fishes were exposed for 96 hours to the test solution. The results showed that under valid static test conditions, the 96 h-LC50 of the test substance to fish (rare minnow, gobiocypris rarus) is 18.3 mg/l with 95% confidence limit of 14.8 mg/l ~ 22.6 mg/l (based on measured concentration).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

3.2 mg/L