[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Oct 2014 to 02 Nov 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

July 26, 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures)

Version / remarks:

September 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Frequency of measurement: At least once a week
- Sampling for measurement: The mixed solution taken out with equal volume from the middle layer of test vessel each test level.

Vehicle:

no

Details on test solutions:

- Preparation of test solution: Addition of the test sample was conducted by content using the density (0.998 g/cm3). After the test sample was added into dilution water in Erlenmeyer flask under the water surface with Eppendorf to produce 21.45 mg/L (nominal), the flask was immediately sealed with a plug without headspace. It was stirred by magnetic stirrer for 24 hours to prepare the stock solution. The prepared stock solution was filled into a bag without headspace. A certain amount of the stock solution adjusted by a metering pump and dilution water adjusted by diluter system was admixed to prepare the test solution of the highest test concentration, and the test solution was diluted with dilution water in a stepwise fashion to prepare each test solution, continuously. The flow rates were checked at regular intervals and their variation throughout exposure duration resulted in less than 10%. In addition, the stock solution was replaced within 4 days after the preparation using newly prepared stock solutions, because it was confirmed that stock solutions were stable for 4 days under the room temperature. At the start of exposure, test solution was clear and colorless. At the end of exposure, test solution was also clear and colorless.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name:
Fathead minnow
- Source:
Cultivated in the test facility
- Feeding during test: Fish after hatching was fed at food satiation with newly hatched Artemia nauplii (< 24 h). Feeding was conducted twice daily. The quantity of feeding was revised based on the background data about the growth once a week. In addition, after the second, third and fourth week, the quantity of feeding was revised appropriately when decrease of fish by death were observed. Feeding on the end of exposure and the day before was not conducted. Residual food and feces in the test vessel were removed at appropriate frequency. The feeding volume is provided in Table 1 in 'Any other information on materials and methods incl. tables'.

ACCLIMATION
- Hatching date (parent fish): February 24, 2014 (age at the start of exposure: seven-month-old)
- Type of acclimation: Flow-through regime
- Dissolved oxygen concentration: ≥ 80% ofair saturation value
- Temperature: 25 ±1.5°C
- Lighting condition: Room light, 16-hour light/8-hour darkness
- Feed: Feed for fry of carp "crumble 2C" (Nippon Formula Feed Mfg.); Newly hatched Artemia nauplii (< 24 h)
- Feeding frequency during acclimation:
Suitable volume without residue food was fed everyday
- Use of medicament for external disinfection: None

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

4 days embryo phase and 28 days post-hatch phase

Hardness:

42.2 - 50.03 mg CaCO3/L.

Test temperature:

25 ± 1.5°C

pH:

7.7 - 8.1

Dissolved oxygen:

7.7 - 8.5 mgO2/L (≥60% ofsaturate concentration)

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0.0062, 0.019, 0.056, 0.17, 0.50 mg/L
- Measured concentrations: N.D. (negative control), 0.00487, 0.0145, 0.0439, 0.118, 0.316 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel:
3 L Glass tank (diameter: 16 cm, depth: 17 cm); The tank has an overflow tube set at approximately 1.8 L level. Air tight transparent plastic lid on test vessel.
- Type: Closed
(with headspace)
- Volume of test solution: Approximately 7.2 L/test level (approx. 1.8 L/test vessel)
- Aeration:
No
- Diluter system: Apparatus to deliver the stock solution of the test sample and dilution water, which were adjusted to a constant flow rate with metering pump and capillary tube respectively, into a mixing container, mix with a magnetic stirrer, and supply the mixed solution continuously to the test vessel as test solution after dividing it into 4 vessels. Diluter system avoided opening area as much as possible.
- Flow rate: Approximately 12 times renewed per day
- No. of organisms per vessel:
80 embryos/test level (20 embryos/test vessel)
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Biomass loading rate:
Not exceeding 0.5 g/L per 24 hours (actual maximum rate: 0.0538 g/l) and not exceeding 5 g/L of solution at any time (actual maximum rate: 0.645 g/L) at anytime
- Operation of study: Test organisms were exposed as soon as possible after the fertilization. Laid eggs were removed from the spawning surface and they were used for study. The embryos were put in stainless steel basket by 20 embryos (80 embryos/test level) at random. The developmental stage of embryo at the start of exposure was morula stage.
- Rotation of test vessel: To minimize the variation of the environmental conditions among the test vessels in each test level, the test vessels were rotated once a week.

TEST MEDIUM
- Source/preparation of dilution water:
Dechlorinated tap water, aerated sufficiently and temperature-controlled, was used. It was confirmed that the concentration of residual chlorine in dilution water was below 0.02 mg/L once a day.

WATER PARAMETERS
- Dissolved oxygen concentration (DO), pH and temperature: All test vessels were measured at least once a week. In addition, continuous temperature was monitored in one test vessel of the control.
- Total hardness: All test vessels were measured at the start and end of exposure.

OTHER TEST CONDITIONS
- Adjustment of pH:
No
- Photoperiod:
16-hour light and 8-hour in dark
- Light intensity:
Room light

EFFECT PARAMETERS MEASURED
- Embryo phase (observation): Hatching and mortality were observed daily. The stereoscopic microscope was used for observation if necessary. Eye formation and presence of developmental abnormity in the developmental process were observed in addition. Dead embryo after the observation was removed as soon as possible. The criterion of the death is shown as follow:
For eggs: Marked loss of translucency and change in colouration, leading to a white opaque
For embryos: Absence of body movement and/or absence of heart-beat
- After hatching (observation): Mortality, behaviour (abnormal respiration, abnormal swimming, impossible to swim, abnormal feeding activity, and so on) and presence of abnormal appearance were observed daily. The stereoscopic microscope was use for observation if necessary. Dead fish was removed after the observation as soon as possible. The criterion of the death is shown as follow:
For larvae and juvenile fish: Immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque colouration of central nervous system and/or lack of reaction to mechanical stimulus.
- Body weight and total length of test organism: The wet body weight and total length of all surviving fish were measured at the end of exposure.
- Appearance of test solution: Appearance of the test solutions was observed at the start and end of exposure.

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

EC10

Effect conc.:

0.074 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

weight

Remarks on result:

other: 95% C.L. 0.050 - 0.098 mg/L

Duration:

32 d

Dose descriptor:

EC50

Effect conc.:

0.318 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

weight

Remarks on result:

other: 95% C.L. 0.236-0.401 mg/L

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.118 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: survival rate after hatching, body weight and total length

Duration:

32 d

Dose descriptor:

LOEC

Effect conc.:

0.316 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: survival rate after hatching, body weight and total length

Details on results:

An overview of the results is provided in Table 3 – Table 7 in ‘Any other information on results incl. tables’.
- Hatchability: Hatchability in control was 98.8%, which meets the criterion for the validity of the test (i.e. more than 70%). For hatchability, no significant difference (p < 0.05) was observed between all exposure levels and control.
- Average of days to hatch: For average of days to hatch, no significant difference (p < 0.05) was observed between all exposure levels and control.
- Rate of morphological abnormality: Morphological abnormality was not observed in all test levels.
- Survival rate after hatching: Survival rate after hatching in control at the end of exposure were 91.2%, which meets the criterion for the validity of the test (i.e. more than 75%). For survival rate after hatching, significant difference (p < 0.01) was observed between exposure level of 0.316 mg/L and control. No significant difference (p < 0.05) were observed between other exposure levels and control.
- Body weight of survival fish at end of exposure: For wet body weight of survival fish at the end of exposure, significant difference (p < 0.01) was observed between exposure level of 0.316 mg/L and control. No significant difference (p < 0.05) was observed between other exposure levels and control.
- Total length of survival fish at end of exposure: For total length, significant difference (p < 0.01) was observed between exposure level of 0.316 mg/L and control. No significant difference (p < 0.05) was observed between other exposure levels and control.
- Observed symptoms and feeding behaviour of test organisms: No symptom was observed at embryo phase in all test levels. Days to eye formation was 2 days after exposure for all survival test organism. Abnormal responses was observed in all test level containing with control after hatching. However the number of the test organisms observed abnormal response was small, without a clear concentration-dependency. Results of observed symptoms were abnormal swimming and abnormal appearance.
- ECx: The calculated EC10 and EC20 for survival rate after hatching were 0.110 mg/L (95% confidence limits: 0.074 - 0.147 mg/L) and 0.173 mg/L (95% confidence limits: 0.120 - 0.225 mg/L), respectively. The calculated EC10, EC20 and EC50 for body weight were 0.074 mg/L (95% confidence limits: 0.050 - 0.098 mg/L), 0.123 mg/L (95% confidence limits: 0.092 - 0.154 mg/L) and 0.318 mg/L (95% confidence limits: 0.236 - 0.401 mg/L), respectively. The calculated EC10 and EC20 for total length were 0.134 mg/L (95% confidence limits: 0.107 - 0.160 mg/L) and 0.264 mg/L (95% confidence limits: 0.216 - 0.312 mg/L), respectively.
- LOEC and NOEC: LOEC and NOEC of the test item for fathead minnow in this study were 0.316 and 0.118 mg/L, respectively.

Reported statistics and error estimates:

The results of this study were estimated based on arithmetic mean of the measured concentrations since the measured concentration of test item in test solution were not maintained within the range of ± 20% of the nominal concentrations during exposure.
- Estimation of LOEC and NOEC: LOEC and NOEC were evaluated by the appropriate statistical methods for following observations and measurements compared with control. Differences were considered to be significant at p
- Estimation of ECx: The data of the ECx about survival rate after hatching (x= 10 and 20), body weight (x=10, 20 and 50) and total length (x=10, 20) and these 95% confidence limits were estimated because the correlation of concentration-response was confirmed. The EC50 of survival rate after hatching and total length were not estimated since these values were over the range of test concentrations. For hatching rate and average of days to hatch, the ECx was not estimated because the correlation of concentration-response was not observed. These ECxs were calculated based on non-linear regression model "model 2: y = a exp (x/b) " described in OECD guidance for statistical analysis (ENV/JM/MONO (2006)18) after the data was corrected by Abbott's formula Non-linear regression analysis was conducted by statistical software (IBM SPSS ver.22.0 for Windows).

Table 3. Hatchability and average of days to hatch

Measured concentration (mg/L)	replicate	Nr. organism	Nr. Of hatch on each exposure day						Total hatched number	Hatchability (%)		Average of days to hatch (day)
			1	2	3	4	5	6		Each vessel	Mean between vessels±SD	Each vessel	Mean between vessels±SD
Control	A	20	0	0	6	14			20	100	98.8 ± 2.5	3.70	3.62 ± 0.19
	B	20	0	0	4	15			19	95.0		3.79
	C	20	0	0	7	13			20	100		3.65
	D	20	0	0	13	7			20	100		3.35
0.00487	A	20	0	0	10	10			20	100	100 ± 0.0	3.50	3.75 ± 0.21
	B	20	0	0	2	18			20	100		3.90
	C	20	0	0	1	19			20	100		3.95
	D	20	0	0	7	13			20	100		3.65
0.0145	A	20	0	0	9	11			20	100	100 ± 0.0	3.55	3.56 ± 0.09
	B	20	0	0	8	12			20	100		3.60
	C	20	0	0	11	9			20	100		3.45
	D	20	0	0	7	13			20	100		3.65
0.0439	A	20	0	0	5	15			20	100	100 ± 0.0	3.75	3.76 ± 0.06
	B	20	0	0	6	14			20	100		3.70
	C	20	0	0	3	17			20	100		3.85
	D	20	0	0	6	13	1		20	100		3.75
0.118	A	20	0	0	5	15			20	100	98.8 ± 2.5	3.75	3.71 ± 0.08
	B	20	0	0	7	13			20	100		3.65
	C	20	0	0	4	16			20	100		3.80
	D	20	0	0	7	12			19	95.0		3.63
0.316	A	20	0	0	5	15			20	100	100 ± 0.0	3.75	3.71 ± 0.13
	B	20	0	0	3	17			20	100		3.85
	C	20	0	0	9	11			20	100		3.55
	D	20	0	0	6	14			20	100		3.70

Blank expresses no embryo lor observation.

Table 4. Rate of morphological abnormality in embryo

Measured concentration (mg/L)	replicate	Nr. organism	Number of morphological abnormalities in embryo on each exposure day						Maximum number	Rate of developmental abnormality (%)
			1	2	3	4	5	6		Each vessel	Mean between vessels±SD
Control	A	20	0	0	0				0	0	0±0
	B	20	0 (1)	0	0				0	0
	C	20	0	0	0				0	0
	D	20	0	0	0				0	0
0.00487	A	20	0	0	0				0	0	0±0
	B	20	0	0	0				0	0
	C	20	0	0	0				0	0
	D	20	0	0	0				0	0
0.0145	A	20	0	0	0				0	0	0±0
	B	20	0	0	0				0	0
	C	20	0	0	0				0	0
	D	20	0	0	0				0	0
0.0439	A	20	0	0	0				0	0	0±0
	B	20	0	0	0				0	0
	C	20	0	0	0				0	0
	D	20	0	0	0	0			0	0
0.118	A	20	0	0	0				0	0	0±0
	B	20	0	0	0				0	0
	C	20	0	0	0				0	0
	D	20	0	0 (1)	0				0	0
0.316	A	20	0	0	0				0	0	0±0
	B	20	0	0	0				0	0
	C	20	0	0	0				0	0
	D	20	0	0	0				0	0

Blank expresses no embryo lor observation.

In parentheses show the number of dead embryo.

Table 5. Survival rate after hatching

Measured concentration (mg/L)	replicate	Nr. organism	Number of fish surviving on each exposure day after hatching				Survival rate at end of exposure (%)
			7	14	21	28	Each vessel	Mean between vessels ± S.D.
Control	A	20	0	0	0		90.0	91.2 ± 2.4
	B	20	0 (1)	0	0		94.7
	C	20	0	0	0		90.0
	D	20	0	0	0		90.0
0.00487	A	20	0	0	0		90.0	92.5 ± 5.0
	B	20	0	0	0		100.0
	C	20	0	0	0		90.0
	D	20	0	0	0		90.0
0.0145	A	20	0	0	0		100.0	95.8 ± 2.5
	B	20	0	0	0		100.0
	C	20	0	0	0		95.0
	D	20	0	0	0		100.0
0.0439	A	20	0	0	0		85.0	93.8 ± 7.5
	B	20	0	0	0		100.0
	C	20	0	0	0		100.0
	D	20	0	0	0	0	90.0
0.118	A	20	0	0	0		100.0	93.7 ± 9.5
	B	20	0	0	0		100.0
	C	20	0	0	0		80.0
	D	20	0	0 (1)	0		94.7
0.316	A	20	0	0	0		65.0	45.8 ± 14.4**
	B	20	0	0	0		50.0
	C	20	0	0	0		30.0
	D	20	0	0	0		50.0

Blank expresses no embryo lor observation.

In parentheses show the number of dead embryo.

Table 6. Body weight and total length of survival fish at end of exposure

Measured concentration (mg/L)	replicate	Individual wet body weight (mg)			Individual total length (cm)
		Each vessel Mean±S.D.	Each vessel CV(%)	CV(%) between replicate	Each vessel Mean±S.D.	Each vessel CV(%)	CV(%) between replicate
Control	A	53.0±27.0	51.0	1.5	1.91±0.37	19.5	1.6
	B	52.6±25.3	48.2		1.92±0.34	17.7
	C	52.9±30.2	57.2		1.85±0.37	19.9
	D	54.4±27.8	51.1		1.88±0.40	21.5
0.00487	A	57.0±24.1	42.4	7.2	1.95±0.31	15.9	3.0
	B	57.3±27.2	47.5		1.87±0.36	19.2
	C	50.7±20.4	40.3		1.86±0.30	16.2
	D	50.1±23.1	46.1		1.81±0.32	17.6
0.0145	A	58.1±26.0	44.7	4.9	1.88±0.34	18.3	1.0
	B	56.8±30.1	52.9		1.88±0.35	18.5
	C	52.1±20.1	38.6		1.89±0.27	14.4
	D	54.1±24.0	44.4		1.85±0.35	19.1
0.0439	A	43.4±21.7	50.1	10.4	1.74±0.32	18.4	4.2
	B	53.4±20.9	39.1		1.89±0.25	13.0
	C	55.5±24.8	44.7		1.91±0.33	17.5
	D	50.9±22.9	45.0		1.86±0.30	16.4
0.118	A	56.7±28.8	50.8	13.4	1.89±0.39	20.5	4.3
	B	54.3±25.1	46.2		1.87±0.32	17.1
	C	42.2±18.0	42.7		1.71±0.28	16.1
	D	46.8±19.7	42.2		1.80±0.27	14.8
0.316	A	23.5±10.6	45.2	12.6**	1.46±0.21	14.7	5.1**
	B	25.2±15.4	61.3		1.41±0.32	22.6
	C	18.7±8.8	47.0		1.30±0.19	14.8
	D	24.2±9.9	40.7		1.44±0.19	13.5

** Significantly different from control at p < 0.01

Table 7. LOEC and NOEC

Measured concentration (mg/L)	Hatchability	Average days of hatch	Rate of morphological abnormality	Survival rate after hatching	Body weight	Total length
0.00487	-	-	-	-	-	-
0.0145	-	-	-	-	-	-
0.0439	-	-	-	-	-	-
0.118	-	-	-	-	-	-
0.316	-	-	-	**	**	**

- No significantly different at p < 0.05

** Significantly different at p< 0.01

 

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'

Conclusions:

In a fish Early Life Stage test in fathead minnow (Pimephales promelas) which was performed in accordance with OECD TG 210, a 32-d EC10 for body weight was determined to be 0.074 mg/L. A NOEC of 0.118 mg/L was established.

Executive summary:

A fish Early Life Stage test was performed in fathead minnow (Pimephales promelas) in accordance with OECD TG 210 and GLP criteria. Fish embryo (20/replicate, 4 replicates/concentration) were exposed to nominal concentrations of 0 (negative control), 0.0062, 0.019, 0.056, 0.17 and 0.50 mg/L under flow-through condition (biomass loading rate ≤0.5 g/L per 24 hours) for 32 days (4 days embryo phase and 28 days post-hatch phase). The test concentrations were analytically verified (measured by GCMS) and determined as ND (negative control), 0.00487, 0.0145, 0.0439, 0.118 and 0.316 mg/L (54.0 – 92.9% of nominal concentrations). The test conditions were 25 ± 1.5°C, pH 7.7 - 8.1 and dissolved oxygen concentration≥ 60% of the air saturation value throughout the test.

After 32 days exposure, the hatchability in control was 98.8%. No significant difference (p < 0.05) on hatchability, average of days to hatch or morphological abnormality was observed between all exposure levels and control. Survival rate after hatching in control at the end of exposure were 91.2%. For survival rate after hatching, wet body weight of survival fish and total length of survival fish, significant difference (p < 0.01) was observed between exposure level of 0.316 mg/L and control. No significant difference (p < 0.05) was observed between other exposure levels and control for those parameters. No symptoms were observed at embryo phase in all test levels. Based on these findings, the 32-day EC10 was determined to be 0.074 mg/L (95% C.L. is 0.050 – 0.098 mg/L) for the total weight of survival fish based on the mean measured concentrations. A NOEC of 0.118 mg/L was established.

Description of key information

A fish Early Life Stage test was performed in fathead minnow (Pimephales promelas) in accordance with OECD TG 210 and GLP criteria. Fish embryo (20/replicate, 4 replicates/concentration) were exposed to nominal concentrations of 0 (negative control), 0.0062, 0.019, 0.056, 0.17 and 0.50 mg/L under flow-through condition (biomass loading rate ≤0.5 g/L per 24 hours) for 32 days (4 days embryo phase and 28 days post-hatch phase). The test concentrations were analytically verified (measured by GCMS) and determined as ND (negative control), 0.00487, 0.0145, 0.0439, 0.118 and 0.316 mg/L (54.0 – 92.9% of nominal concentrations). The test conditions were 25 ± 1.5°C, pH 7.7 - 8.1 and dissolved oxygen concentration≥ 60% of the air saturation value throughout the test. After 32 days exposure, the hatchability in control was 98.8%. No significant difference (p < 0.05) on hatchability, average of days to hatch or morphological abnormality was observed between all exposure levels and control. Survival rate after hatching in control at the end of exposure were 91.2%. For survival rate after hatching, wet body weight of survival fish and total length of survival fish, significant difference (p < 0.01) was observed between exposure level of 0.316 mg/L and control. No significant difference (p < 0.05) was observed between other exposure levels and control for those parameters. No symptoms were observed at embryo phase in all test levels. Based on these findings, the 32-day EC10 was determined to be 0.074 mg/L (95% C.L. is 0.050 – 0.098 mg/L) for the total weight of survival fish based on the mean measured concentrations. A NOEC of 0.118 mg/L was established.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Effect concentration:

0.074 mg/L