Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

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:

The study was conducted between 28 January 2015 and 22 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 236 using a Fish Embryo Acute Toxicity (FET) method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, Test No. 236: Fish Embryo Acute Toxicity (FET) Test; adopted 26 July 2013.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analysis of the test item concentrations, duplicate samples from the test media of all test concentrations and from the control were taken at the start and the end of two test medium renewal periods (Day 0 to Day 1 and Day 3 to Day 4). One of the duplicate samples was incubated in polystyrene multiwell test plates with wells of big volume in parallel to the test. The seconds sample was incubated in glass vessels as a back up.

Storage of the samples
All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

Vehicle:

no

Details on test solutions:

Due to the low solubility of the test item in test water, a dispersion with the loading rate of 100 mg/L was prepared prior to the start of the test and prior to each test medium renewal by dispersing the test item in test water:

Day 0: 111.8 mg in 1120 mL test water
Day 1: 100.5 mg in 1000 mL test water
Day 2: 108.0 mg in 1075 mL test water
Day 3: 107.5 mg in 1075 mL test water

This preparation was supported by intense stirring by magnetic stirrers over 24 hours at room temperature in the dark to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used. As a precaution, stirring was performed in completely closed bottles to avoid possible losses of the test item by evaporation.

The stirring period of 24 hours was selected according to the results of a GLP study with the same test item conducted at Harlan Laboratories Ltd., Shardlow, UK (Harlan Study Number 41401231).

After the 24-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Whatman, Type NC20, pore size 0.2 µm) after preconditioning of the filter with about 200 mL of the dispersion. The negative pressure of the filtration unit was reduced as far as possible as a precaution to avoid possible losses of the test item by evaporation during filtration. The undiluted filtrate was used for the preparation of the test media. For this preparation, the filtrate was diluted with test water. The test media were prepared prior to the start of the test (= start of exposure) and prior to each test medium renewal.

The test method is based on the OECD series on testing and assessment No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000 [OECD, 2000].

At each test medium renewal date, the 3,4-dichloroaniline solution was prepared by diluting nominal 50 mg in 500 mL test water followed by a dilution step of 1:25 with test water.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The study was performed with newly fertilized eggs of zebra fish, Danio rerio (Hamilton-Buchanan 1822, Teleostei, Cyprinidae). The origin of the strain of zebra fish is West Aquarium GmbH, 37431 Bad Lauterberg / Germany. At Harlan Laboratories, a brood batch of at least 200 individuals of this strain was held.

The parental fish were held in an aquarium in reconstituted water (= culturing water with the same water quality as described). A 16 hour light to 8 hour dark photoperiod with a 30 minute transition period was applied. The fish were fed with a commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49304 Melle / Germany) and brine shrimps (Artemia salina).

The brood batch was regularly visually checked for abnormal behavior, diseases or mortality. No visible abnormalities were observed in the fish during three months prior to test start and no medication was applied.

Glass dishes covered with a mesh were placed on the bottom of the aquarium one day prior to test start for collecting the newly fertilized eggs for the test. The mesh separated fertilized eggs (which sank to the bottom into the glass dishes) from the parent fish. Spawning started in the morning on Day 0 after turning on the light. The eggs were exposed to the test media within approximately 60 minutes after fertilization.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

The water temperature were measured at the start and the end of the test in the control and all test concentrations in the test medium vessels (used for incubation of analytical stability samples) incubated in the water bath under the same conditions as in the test.
The water temperature at the start and the end of the exposure period was in the range of 26.2 to 26.7°C.

pH:

The pH values were measured at the start and the end of the test in the control and all test concentrations in the test medium vessels (used for incubation of analytical stability samples) incubated in the water bath under the same conditions as in the test.

Dissolved oxygen:

The dissolved oxygen concentrations were measured at the start and the end of the test in the control and all test concentrations in the test medium vessels (used for incubation of analytical stability samples) incubated in the water bath under the same conditions as in the test.

Nominal and measured concentrations:

The following nominal concentrations were tested: The dilutions 1:22, 1:46, 1:100, 1:220, and 1:460 of an undiluted filtrate with a loading rate of 100 mg/L.

Details on test conditions:

The test was conducted in disposable polystyrene multi-well test plates (24 wells per plate with 3 mL filling capacity each). The wells were nearly completely filled (with a minimal headspace) and covered with the corresponding polystyrene lid to reduce the loss of water by evaporation.

The multi-well plates were labeled with the study number and all necessary additional information to ensure unique identification.

Experimental Conditions
The multi-well plates were placed into a temperature regulated water bath to keep the water temperature in the plates constant during the test period. The multi-well plates were preconditioned for 24 hours with the test media before start of the test. During the test, one and the same multi-well plate was used for each renewal period.
The test media were not aerated. A 16-hour light period (with a 30 minute transition period) was applied, with a light intensity at light period of approximately 560 Lux. The test duration was 96 hours.


Study Design
At the start of the test (Day 0), 20 viable, pre-selected eggs, were transferred to the test wells of each treatment (one egg per well and one plate per treatment). Additionally, four eggs for each treatment were used as an internal plate control.

The eggs were taken from the culturing water and were shortly placed into a volume of the corresponding test medium and were immediately thereafter transferred into the wells of the corresponding treatment. This was necessary in order to not dilute the test media in the wells with culturing water.

The eggs used were not older than 60 min post fertilization before being placed into the test media.

The following nominal concentrations were tested: The dilutions 1:22, 1:46, 1:100, 1:220, and 1:460 of an undiluted filtrate with a loading rate of 100 mg/L. Additionally, a control was tested in parallel. The test concentrations were based on the results of a range finding test and a pre-experiment to the test item dosage procedure.

A positive control with 20 eggs at a fixed concentration of 4.0 mg/L 3,4-dichloroaniline was performed in addition to the main test.


Evaluations
Embryonic Development
The embryonic development was observed for each treatment at the start of the test and once each day during the test period. Apical observations performed on each tested embryo included coagulation of embryos, lack of somite formation, non-detachment of tail, and lack of heart beat.

Percent of hatching success was calculated by dividing the number of hatched larvae by the number of inserted eggs.

The mortality rate was calculated by dividing the number of dead embryos plus the number of dead larvae by the number of inserted eggs.

The development rate was calculated for all treatments with surviving larvae. The mean hatching time represents the mean time span between the start of the test (Day 0) and the hatching of the experimental cohort of larvae. The development rate is the reciprocal of the hatching time (unit: 1/day) and represents that portion of larvae hatching per day. For the statistical evaluation the number of hatched larvae observed on inspection Day x were assumed to have hatched at the mean of the time interval between Day x and Day x-l (l = length of the inspection interval = 1 day). The mean of the development rate per treatment was calculated.
The NOEC and LOEC for hatching rate of larvae and mortality of embryos/larvae was determined by a Fisher’s exact binomial test (one-sided greater, α = 0.05) [Sachs, 1984]. The LC10 value for development rate was calculated by Probit Analysis [Finney, 1971; Davies, 1971] as a representative for the NOEC.

The LC50 values for mortality of embryos/larvae and the 95%-confidence intervals after 48 and 96 hours were calculated by Weibull Analysis [Christensen, 1984 ].

The statistical calculations were performed using ToxRat Professional,ToxRat Solutions GmbH, Version 2.10.05 (released 20.02.2010). Calculations were based on mean measured test item concentrations and were corrected by control mortality.

The LC0 and LC100 values were determined directly from the raw data.


Calculation of Mean Measured Test Item Concentration
For each test medium renewal period, the mean concentration was calculated as geometric mean of the test item concentrations measured at the start and the end of the test medium renewal period. From the values obtained, the mean measured test item concentration during the test period was calculated as arithmetic mean.

Calculation formula of mean measured test item concentration:

(√((Day0)*(day 1)) + √((Day3)*(Day 4))) / n

n: number of analyzed renewal intervals

Water Quality Criteria
The appearance of the test media were observed each working day.

Reference substance (positive control):

yes

Remarks:

3,4-dichloroaniline

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks on result:

other: No adverse effects of the test item on development of larvae, hatching success, and mortality were determined.

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Based on adverse effects on hatching success, development rate, and mortality of the embryos and larvae.

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks on result:

other: 95%-confidence limits of 1.1 and 4.4 mg/L for embryos and larvae

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks on result:

other: 95%-confidence limits of 0.85 and 1.6 mg/L for embryos and larvae

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 1.35 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: The 96-hour LC100 was greater than the highest concentration tested

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Hatching Success
In the control one living embryo was found not to have hatched and one embryo was dead at the end of the test on Day 4 post fertilization. Thus, the hatching success in the control was determined to be 90% and was sufficiently high under the test conditions to fulfill the validity criterion of the test guideline for hatching success (≥80%).

In the control and up to and including the highest test concentrations of 1.35 mg/L (mean measured), first larvae had hatched on Day 3.

At the test concentrations up to and including 0.29 mg/L, hatching success was in the range of 85 to 90%, corresponding to 94 to 100% of the control value. At the test concentration of 0.63 and 1.35 mg/L, hatching success was clearly reduced to 55 and 40%, corresponding to 61 and 44% of the control value. The statistical evaluation (Fisher’s exact binomial test, one-sided greater, α = 0.05) showed a statistically significant reduction only at the test concentration of 1.35 mg/L. However, the clearly reduced hatching success at 0.63 mg/L was considered to be a relevant part of the concentration effect curve and therefore the NOEC was determined to be 0.29 mg/L. The LOEC was 0.63 mg/L.


Development Rate of Embryos
The development rate of the embryos at all test concentrations up to and including 0.29 mg/L was in the range of 97 to 107% of the control value. At 0.63 and 1.35 mg/L, the development rate was reduced to 57 and 38% of the control value. The EC10 for the development rate was calculated as a representative of the NOEC and was 0.27 mg/L (Probit Analysis, one-sided smaller, α = 0.05).

Therefore, the test concentration of 0.29 mg/L was determined to be the NOEC. The LOEC was 0.63 mg/L.


Mortality of Embryos and Larvae
In the control, one living larvae had not hatched until Day 4. One larvae was found to be dead and therefore the mortality of the embryos and larvae in the control was 5% and was sufficiently high to meet the validity criterion of the test guideline (≥90% survival).

At the test concentrations up to and including 0.29 mg/L, mortality was in the range of 5 to 10%, corresponding to a survival rate of 95 to 100% compared to the control value. At the test concentration of 0.63 and 1.35 mg/L, mortality of embryos and larvae was clearly increased to 35 and 60%, corresponding to a survival rate 68 and 42% of the control value. The statistical evaluation (Fisher’s exact binomial test, one-sided greater, α = 0.05) showed a significant reduction only at the test concentration of 1.35 mg/L. However, the clearly increased mortality at 0.63 mg/L was considered to be a relevant part of the concentration effect curve and therefore the NOEC was determined to be 0.29 mg/L. The LOEC 0.63 was mg/L.

The 48-hour LC50 for embryos and larvae was calculated to be 1.6 mg/L, with 95%-confidence limits of 1.1 and 4.4 mg/L.

The 96-hour LC50 for embryos and larvae was calculated to be 1.1 mg/L, with 95%-confidence limits of 0.85 and 1.6 mg/L.

The 96-hour LC100 was higher than the highest concentration tested (>1.35 mg/L). The 96-hour LC0 was assigned to the test concentration of 0.29 mg/L.

At the positive control, the mortality was 100%, fulfilling the requirement of the test guideline.
The mortality in the internal plate controls was in maximum one dead embryo/larvae per plate, fulfilling the requirement of the test guideline.


Apical Endpoints
At the test concentrations up to and including 0.29 mg/L and at the control, all larvae which survived until the end of the test were healthy and showed normal development. There was no lack of somite formation and no non-detachment of the tail, i.e. somite formation and detachment of the tail was observed first at the observation on Day 1. Heartbeat of larvae was observed at these test concentrations starting from Day 2. Embryos and larvae found to be dead at test concentrations up to and including 0.29 mg/L and at the control were considered as natural mortality.

At the test concentrations of 0.63 and 1.35 mg/L the embryos and larvae found to be dead at the different treatments showed coagulation, or deformation, lack of development or were undergoing decomposition. The surviving embryos visually appeared to be not affected by the test item and somite formation, detachment of the tail and heartbeat was observed.

Results with reference substance (positive control):

At the positive control, the mortality was 100%, fulfilling the requirement of the test guideline.

Reported statistics and error estimates:

The development rate was calculated for all treatments with surviving larvae. The mean hatching time represents the mean time span between the start of the test (Day 0) and the hatching of the experimental cohort of larvae. The development rate is the reciprocal of the hatching time (unit: 1/day) and represents that portion of larvae hatching per day. For the statistical evaluation the number of hatched larvae observed on inspection Day x were assumed to have hatched at the mean of the time interval between Day x and Day x-l (l = length of the inspection interval = 1 day). The mean of the development rate per treatment was calculated.
The NOEC and LOEC for hatching rate of larvae and mortality of embryos/larvae was determined by a Fisher’s exact binomial test (one-sided greater, α = 0.05) [Sachs, 1984]. The LC10 value for development rate was calculated by Probit Analysis [Finney, 1971; Davies, 1971] as a representative for the NOEC.

The LC50 values for mortality of embryos/larvae and the 95%-confidence intervals after 48 and 96 hours were calculated by Weibull Analysis [Christensen, 1984 ].

The statistical calculations were performed using ToxRat Professional,ToxRat Solutions GmbH, Version 2.10.05 (released 20.02.2010). Calculations were based on mean measured test item concentrations and were corrected by control mortality.
The LC0 and LC100 values were determined directly from the raw data.

Sublethal observations / clinical signs:

Analytical Results

The calculated mean measured concentrations (mean of the two renewal periods) of IFF 215 (Floriane) at the start and the end of the renewal periods are shown in the table below. During the test medium renewal periods of 24 hours a decrease of the test item concentration in the test media was determined. At the end of the test medium renewal periods, 59 to 74% of the initially mean measured concentrations were found.

 

The mean measured concentrations during the test period was calculated as the arithmetic mean of the two geometric means determined for the two test medium renewal periods for each test concentration:

 

Test concentration / Dilution*	Measured concentrations first renewal period [mg/L]		Measured concentrations second renewal period [mg/L]		Mean measured concentration during exposure period
	Start	End	Start	End	[mg/L]
1:460	0.077	0.053	0.080	0.053	0.065
1:220	0.16	0.10	0.16	0.12	0.13
1:100	0.34	0.21	0.39	0.26	0.29
1:46	0.76	0.45	0.85	0.54	0.63
1:22	1.6	0.93	1.8	1.2	1.35

*: Dilutions of an undiluted filtrate with a loading rate of 100 mg/L

SD: Standard Deviation

 

The biological results were related to the mean measured test item concentrations.

Water Quality Criteria

At the beginning and end of the test period, the dissolved oxygen concentration in the test medium and control was at least 7.9 mg/L, corresponding to 97% oxygen concentration. The pH values of the test medium and control were between 7.5 and 7.7. The water temperature during the test was between 26.2 and 26.7 °C.

Validity Criteria

The test was considered to be valid since all relevant validity criteria have been fulfilled.

HatchingSuccess 

Mean measured test item concentration (mg/L)	Larvae hatched / viable eggs* (at test start 20 eggs were inserted)				No. of larvae hatched	No. of living embryos (not hatched)	No. of dead embryos	Hatching rate (%)	% of control
	Day 1	Day 2	Day 3	Day 4
Control	0 / 20	0 / 20	10 / 10	18 / 1	18	1	1	90	-
0.065	0 / 20	0 / 20	14/5	18 / 1	18	1	1	90	100
0.13	0 / 20	0 / 20	8 / 12	18 / 1	18	1	1	90	100
0.29	0 / 19	0 / 18	11 / 7	17 / 1	17	1	2	85	94
0.63	0 / 19	0 / 16	4 / 9	11 / 2	11	2	7	55	61
1.35	0 / 15	0 / 12	1 / 7	8 / 0	8	0	12	40	44
3,4-DCA**	0 / 14	0 / 6	0 / 0	0 / 0	0	0	20	0	0

*: Dead eggs are not included in this counting.

**: Reference Item 3,4-Dichloroaniline at a concentration of 4.0 mg/L.

 

 

Development Rate of Embryos

Mean measured test item concentration (mg/L)	No. of larvae hatched					Development rate (1 / day)	% of control
	Day 1	Day 2	Day 3	Day 4	Sum
Control	0	0	10	8	18	6.286	-
0.065	0	0	14	4	18	6.473	107
0.13	0	0	8	10	18	6.057	96
0.29	0	0	11	6	17	6.114	97
0.63	0	0	4	7	11	3.600	57
1.35	0	0	1	7	8	2.400	38
3,4-DCA**	0	0	0	0	0	0.000	0

**: Reference Item 3,4-Dichloroaniline at a concentration of 4.0 mg/L.

 

Cumulative Mortality of Embryos and Larvae

Mean measured test item concentration (mg/L)	Mortality of embryos and larvae at the observation days					Mortality rate (%)	Survival in % of control (%)
	Day 1	Day 2	Day 3	Day 4	Sum
Control	0	0	0	1	1	5
0.065	0	0	0	1	1	5	100
0.13	0	0	0	1	1	5	100
0.29	1	1	0	0	2	10	95
0.63	1	3	3	0	7	35	68
1.35	5	3	4	0	12	60	42
3,4-DCA**	6	8	6	0	20	100	0

**: Reference Item 3,4-Dichloroaniline at a concentration of 4.0 mg/L.

Water Temperature in the Treatments

Mean measured test item concentration [mg/L]	Exposure time
	0 h	24 h		48 h			72 h		96 h
	new	old	new	old	new	old		new	old
Control	26.7	26.4	26.7	26.3	26.5	26.2		26.7	26.3
0.065	26.7	26.4	26.7	26.3	26.5	26.2		26.7	26.3
0.13	26.7	26.4	26.7	26.3	26.5	26.2		26.7	26.3
0.29	26.7	26.4	26.7	26.3	26.5	26.2		26.7	26.3
0.63	26.7	26.4	26.7	26.3	26.5	26.2		26.7	26.3
1.35	26.7	26.4	26.7	26.3	26.5	26.2		26.7	26.3
3,4-DCA	26.7	26.4	26.7	26.3	26.5	26.2		--	--

--: Not determined since all embryos were dead

 

 

Dissolved Oxygen Concentrations in the Treatments

Mean measured test item concentration [mg/L]	Exposure time
	0 h	24 h		48 h			72 h		96 h
	new	old	new	old	new	old		new	old
Control	8.0	8.1	8.0	7.9	8.0	8.1		7.9	8.2
0.065	8.5	8.1	8.0	7.9	8.0	8.0		7.9	8.0
0.13	8.5	8.1	8.0	7.9	8.0	8.0		7.9	8.1
0.29	8.5	8.1	8.0	7.9	8.0	8.0		7.9	8.1
0.63	8.5	8.1	8.0	7.9	8.0	8.0		7.9	8.1
1.35	8.4	8.1	8.0	7.9	7.9	8.0		7.9	8.2
3,4-DCA	8.4	8.1	8.0	7.9	8.0	8.1		--	--

--: Not determined since all embryos were dead

 

pH Values in the Treatments

Mean measured test item concentration [mg/L]	Exposure time
	0 h	24 h		48 h			72 h		96 h
	new	old	new	old	new	old		new	old
Control	7.6	7.6	7.7	7.7	7.6	7.6		7.6	7.7
0.065	7.6	7.6	7.7	7.7	7.6	7.5		7.7	7.7
0.13	7.6	7.6	7.7	7.7	7.6	7.5		7.7	7.7
0.29	7.6	7.6	7.7	7.7	7.6	7.5		7.7	7.7
0.63	7.6	7.6	7.7	7.7	7.6	7.6		7.7	7.7
1.35	7.5	7.5	7.7	7.7	7.6	7.6		7.7	7.7
3,4-DCA	7.6	7.5	7.7	7.7	7.6	7.6		--	--

--: Not determined since all embryos were dead

Validity criteria fulfilled:

yes

Conclusions:

Summarizing the results from the different test parameters assessed, the NOEC for embryos and larvae of zebra fish was determined to be 0.29 mg/L, since no adverse effects of the test item IFF 215 (Floriane) on development of larvae, hatching success, and mortality were determined.

The LOEC was determined to be 0.63 mg/L due to distinct adverse effects on hatching success, development rate, and mortality of the embryos and larvae.

The 96-hour LC50 for embryos and larvae was calculated to be 1.1 mg/L, with 95%-confidence limits of 0.85 and 1.6 mg/L.
The 96-hour LC100 was higher than the highest concentration tested (>1.35 mg/L).
The 96-hour LC0 was assigned to the test concentration of 0.29 mg/L.

Executive summary:

The toxicity of the test item, IFF 215 (Floriane), on embryos and larvae of zebra fish was investigated in a fish embryo acute toxicity test according to the OECD Guideline for Testing of Chemicals,Test No. 236 using a Fish Embryo Acute Toxicity (FET) method.

The NOEC for embryos and larvae of zebra fish was determined to be 0.29 mg/L, since no adverse effects of the test item on development of larvae, hatching success, and mortality were determined.

 

The LOEC was determined to be 0.63 mg/L due to distinct adverse effects on hatching success, development rate, and mortality of the embryos and larvae.

The 48-hour LC₅₀for embryos and larvae was calculated to be 1.6 mg/L, with 95%-confidence limits of 1.1 and 4.4 mg/L.

The 96-hour LC₅₀for embryos and larvae was calculated to be 1.1 mg/L, with 95%-confidence limits of 0.85 and 1.6 mg/L.

The 96-hour LC₁₀₀was greater than the highest concentration tested (>1.35 mg/L).

The 96-hour LC₀was assigned to the test concentration of 0.29 mg/L.

Description of key information

The toxicity of the test item, IFF 215 (Floriane), on embryos and larvae of zebra fish was investigated in a fish embryo acute toxicity test according to the OECD Guideline for Testing of Chemicals,Test No. 236 using a Fish Embryo Acute Toxicity (FET) method. 
The NOEC for embryos and larvae of zebra fish was determined to be 0.29 mg/L, since no adverse effects of the test item on development of larvae, hatching success, and mortality were determined.
 
The LOEC was determined to be 0.63 mg/L due to distinct adverse effects on hatching success, development rate, and mortality of the embryos and larvae.
The 48-hour LC50for embryos and larvae was calculated to be 1.6 mg/L, with 95%-confidence limits of 1.1 and 4.4 mg/L.
The 96-hour LC50for embryos and larvae was calculated to be 1.1 mg/L, with 95%-confidence limits of 0.85 and 1.6 mg/L.
The 96-hour LC100was greater than the highest concentration tested (>1.35 mg/L).
The 96-hour LC0was assigned to the test concentration of 0.29 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.1 mg/L