(octahydro-4,7-methano-1H-indenyl)methyl acrylate

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:

2018-06-21 to 2018-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

There was a deviation from the study plan:
At the analysis of 2018-07-10, the concentration of the saturated solution was quantitatively analyzed with ion 148 due to wrong selection of quantitative ion, which deviated from the quantitative analysis method in study plan: select ion 80 as quantitative ion. Because the difference of two results was slight (22.2404 mg/L with selected ion 148 and 22.8415 mg/L with selected ion 80) and the concentrations of test solutions was finally quantitatively analyzed with ion 80, the deviation did not affect the quality or integrity of the study.

Deviations:

yes

Remarks:

See "Version / remarks"

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Method: GC/MS/MS
Analysis frequency: in 0, 48 hour fresh solutions and 48, 96 hour old solutions.
For the test groups of 63.2%SS, 79.5%SS and 100%SS, all fish were dead after 24h, therefore, to characterize the exposure concentrations truer, measurement of test item concentration in old solution of 63.2%SS, 79.5%SS and 100%SS was carried out at 24 hour.

Vehicle:

no

Details on test solutions:

See "any other information on materials and methods incl. tables".

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

See "any other information on materials and methods incl. tables".

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

180 - 190 mg/L as CaCO3

Test temperature:

22.4 - 23.2 °C (first definitive test)
22.8 - 23.4 °C (second definitive test)

pH:

See "any other information on results incl. tables".

Dissolved oxygen:

See "any other information on results incl. tables".

Nominal and measured concentrations:

For the nominal concentrations of 1.69, 2.13, 2.68, 3.38 mg/L and 40%SS, 50.3%SS, 63.2%SS, 79.5%SS and 100%SS, the mean exposure concentrations were 1.059, 1.194, 1.546, 1.948, 2.175, 2.539, 3.152, 5.255 and 7.795 mg/L.

Details on test conditions:

See "any other information on materials and methods incl. tables".

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

See "any other information on results incl. tables".

Sublethal observations / clinical signs:

Table 3. Water Quality and Physicochemical Measurements in the First Definitive Test

Concentration level	Items	0h (Fresh)	24h (Old)	48h (Old)	48h (Fresh)	72h (Old)	96h (Old)
Control	pH	7.66	7.28	7.15	7.49	7.07	7.05
	D.O.	97.5	83.8	78.6	96.5	84.4	82.5
40%SS	pH	7.78	7.20	7.20	7.57	7.18	7.11
	D.O.	96.2	84.1	79.2	94.3	86.9	81.9
50.3%SS	pH	7.78	7.29	7.18	7.58	7.26	7.19
	D.O.	95.3	83.2	78.1	93.1	87.4	83.0
63.2%SS	pH	7.77	7.19	/	/	/	/
	D.O.	94.2	82.4	/	/	/	/
79.5%SS	pH	7.78	7.27	/	/	/	/
	D.O.	93.1	81.3	/	/	/	/
100%SS	pH	7.78	7.19	/	/	/	/
	D.O.	92.3	82.2	/	/	/	/
Total hardness:	190 mg/L (as CaCO3)
Temperature:	22.4 ~ 23.2^oC

Note: D.O., dissolved oxygen, %ASV.

Table 4. Water Quality and Physicochemical Measurements in the Second Definitive Test

Nominal Concentration (mg/L)	Items	0h (Fresh)	24h (Old)	48h (Old)	48h (Fresh)	72h (Old)	96h (Old)
Control	pH	7.94	7.60	7.31	7.66	7.43	7.28
	D.O.	95.0	75.2	74.9	95.8	75.6	72.0
1.69	pH	8.14	7.69	7.41	7.80	7.52	7.31
	D.O.	95.2	73.5	73.2	99.6	76.3	75.2
2.13	pH	8.18	7.69	7.42	7.79	7.53	7.30
	D.O.	94.8	74.0	73.0	99.5	75.5	73.4
2.68	pH	8.21	7.70	7.37	7.87	7.57	7.32
	D.O.	94.9	77.0	72.5	98.9	76.3	74.3
3.38	pH	8.22	7.71	7.39	7.99	7.63	7.35
	D.O.	95.6	76.2	70.5	98.5	77.0	76.7
Total hardness:	180 mg/L (as CaCO3)
Temperature:	22.8 ~ 23.4^oC

Note: D.O., dissolved oxygen, %ASV.

 

Table 5. Cumulative Mortality and Toxic Signs in the First Definitive Test

Concentration level	Geometric mean concentration (mg/L)	Initial Population	Observation Time (h)	Cumulative Dead Number	Mortality rate (%)	Toxic signs^1
Control		< LOQ		7		3		0		0		7A
	24		0		0	7A
	48		0		0	7A
	72		0		0	7A
	96		0		0	7A
40%SS	2.175	7	3	0	0	7F
			24	0	0	7F
			48	0	0	7F
			72		2		28.6		4F1FBL
				96		7		100		/
50.3%SS		2.539		7		3		0		0		7F
			24			0		0		7F
			48			0		0		7F
			72			5		71.4		2F
			96			7		100		/
63.2%SS		3.152		7		3		0		0		7F
			24			7		100		/
79.5%SS		5.255		7		3		0		0		4F3FBL
			24			7		100		/
100%SS		7.795		7		3		5		71.4		2FB
			24			7		100		/

Note: 1. A: no visible toxic signs, B: erratic swimming, F: discoloration, L: body overturning, /:no observation due to 100 % mortality.

Table 6. Cumulative Mortality and Toxic Signs in the Second Definitive Test

Nominal Concentration (mg/L)	Geometric mean concentration (mg/L)	Initial Population	Observation Time (h)	Cumulative Dead Number	Mortality rate (%)	Toxic signs^1
Control	< LOQ	7	3	0	0	7A
			24	0	0	7A
			48	0	0	7A
			72	0	0	7A
			96	0	0	7A
1.69	1.059	7	3	0	0	7A
			24	0	0	7A
			48	0	0	7A
			72	0	0	7A
			96	0	0	7A
2.13	1.194	7	3	0	0	7A
			24	0	0	7A
			48	0	0	7A
			72	0	0	7A
			96	0	0	7A
2.68	1.546	7	3	0	0	7A
			24	0	0	6A1F
			48	0	0	7A
			72		1		14.3		4A2F
				96		1		14.3		4A2F
3.38		1.948		7		3		0		0		4A3F
			24			0		0		4A3F
			48			0		0		4A3F
			72			2		28.6		3F2BF
			96			4		57.1		3F

Note: 1. A: no visible toxic signs, B: erratic swimming, F: discoloration.

Appendix1. Table 5. Concentrations of Test Item in the Exposure Solutionin the First Definitive Test

Concentration level	Sample concentration (mg/L)					Arithmetic mean concentration in fresh media (mg/L)	Arithmetic mean concentration in old media (mg/L)	Old/Fresh
	2018-6-25	2018-6-26	2018-6-27	2018-6-27	2018-6-29
	0h-Fresh	24h-Old	48h-Old	48h-Fresh	96h-Old
Control	<LOQ	/	<LOQ	<LOQ	<LOQ	/	/	/
40%SS	3.6556	/	0.6076	4.8620	1.6819	4.259	1.145	26.9%
0.3%SS	4.8051	/	0.6285	5.7827	1.9296	5.294	1.279	24.2%
63.2%SS	5.5421	1.7924	/	/	/	5.542	1.792	32.3%
79.5%SS	6.5801	4.1961	/	/	/	6.580	4.196	63.8%
100%SS	10.6850	5.6863	/	/	/	10.685	5.686	53.2%
Concentration level	Geometric mean concentration of each renewal period (24 or 48h) (mg/L)					Mean exposure concentration over the whole period (mg/L)
	0-24/48 hour			48-96 hour
40%SS	1.490			2.860		2.175
50.3%SS	1.738			3.340		2.539
63.2%SS	3.152			/		3.152
79.5%SS	5.255			/		5.255
100%SS	7.795			/		7.795

Note:For the test groups of 63.2%SS, 79.5%SS and 100%SS, all fish were dead at 24 h, therefore, measurement was carried out only at 0h-Fresh and24h-Old.

<LOQ,the response in control sample at the retention time of the analyte was <30% of LOQ and the result was expressed as<LOQ.

Appendix1. Table 6. Concentrations of Test Item in the Exposure Solution in the Second Definitive Test

Nominal concentration (mg/L)	Sample concentration (mg/L)				Arithmetic mean concentration in fresh media (mg/L)	Arithmetic mean concentration in old media (mg/L)	Old/Fresh
	2018-7-10	2018-7-12	2018-7-12	2018-7-14
	0h-Fresh	48h-Old	48h-Fresh	96h-Old
Control	*0.3717	<LOQ	<LOQ	<LOQ	/	/	/
1.69	0.9978	1.0408	1.7934	0.6726	1.396	0.857	61.4%
2.13	1.1253	1.0940	2.0475	0.7970	1.586	0.946	59.6%
2.68	1.2930	1.2086	2.9230	1.1592	2.108	1.184	56.2%
3.38	1.6203	1.4872	3.6864	1.4890	2.653	1.488	56.1%
Nominal concentration (mg/L)	Geometric mean concentration of each renewal period (48h) (mg/L)				Mean exposure concentration over the whole period (mg/L)
	0-48 hour		48-96 hour
1.69	1.019		1.098		1.059
2.13	1.110		1.277		1.194
2.68	1.250		1.841		1.546
3.38	1.552		2.343		1.948

Note: *, the control sample may be contaminated during sampling or pretreatment.

<LOQ,the response in control sample at the retention time of the analyte was <30% of LOQ and the result was expressed as<LOQ.

RESULTS AND CONCLUSIONS

Physicochemical Measurements in the Definitive Test

The results of pH value, dissolved oxygen, water total hardness and temperature are shown in Table 3 and 4.

In the first definitive test, the determined values of pH value, dissolved oxygen and temperature were 7.05 ~ 7.78, 78.1 ~ 97.5%ASV and 22.4 ~ 23.2^oC, respectively. In the second definitive test, the determined values of pH value, dissolved oxygen and temperature were 7.28 ~ 8.22, 70.5 ~ 99.6%ASV and 22.8 ~ 23.4^oC, respectively.

Dissolved oxygen and temperature were in the allowed range specified in ‘Conditions of exposure’. pH were within the allowed range: 6.0-8.5.

Verification of Test Concentration in the Definitive Test

The concentrations of test item were determined in samples of test media by GC/MS/MS method. The determined concentrations in old solution were not maintained in the range of 80% ~ 120% of initial determined concentrations, the mean exposure concentration over the whole period was calculated from the geometric mean concentration of each renewal period. For the nominal concentrations of 1.69, 2.13, 2.68, 3.38 mg/L and 40%SS, 50.3%SS, 63.2%SS, 79.5%SS and 100%SS, the mean exposure concentrations were 1.059, 1.194, 1.546, 1.948, 2.175, 2.539, 3.152, 5.255 and 7.795 mg/L.

See Appendix 1 Table 5 and 6.

Toxicity Signs in the Definitive Test

During the 96-hour exposure period, no mortality and other visible toxicity signs were observed in the control group and the measured concentrations of 1.059,1.194 mg/L, the fish exposed to the measured concentrations of 1.546, 1.948 mg/L showed mortality and toxicity signs such as discoloration and erratic swimming, 100% mortality occurred in the measured concentrations of2.175, 2.539, 3.152, 5.255 and 7.795 mg/L.

The results are shown in Table5 and 6.

Conclusion

According to the results and based onthe geometric mean concentration,the96-hoursmedian lethal concentration (LC₅₀) (with 95% confidence limits)of the test itemtoZebra fishwas calculated by theTrimmed Spearman-Karber (TSK) method (EPA, VERSION 1.5) and as followed:

Test duration [hours]	LC50 (mg/L)	95 % lower confidence limit (mg/L)	95 % upper confidence limit (mg/L)
96	1.80	1.65	1.97
LC0(mg/L)= Highest test item concentration with 0 % mortality after 96 hours	1.194
LC100(mg/L)= Lowest test item concentration with 100 % mortality after 96 hours	2.175

Validity of the test

Parameter/Criterion	Results		Conclusion
	First definitive test	Second definitive test
Mortality in the control group: =1 fish at the end of the test	0 dead fish	0 dead fish	The test was considered as valid.
Dissolved oxygen concentration in test solutions: =60% of ASV during the test period	78.1 ~ 97.5%ASV	70.5 ~ 99.6%ASV

Validity criteria fulfilled:

yes

Conclusions:

According to the results and based on the geometric mean concentration, the 96-hours median lethal concentration (LC50) (with 95% confidence limits) of the test item to Zebra fish was calculated by the Trimmed Spearman-Karber (TSK) method (EPA, VERSION 1.5) and as follows:
96h-LC50 = 1.80 mg/L (95% confidence interval: 1.65 – 1.97 mg/L)
96h-LC0 = 1.194 mg/L
96h-LC100 = 2.175 mg/L

Executive summary:

A fish acute toxicity study was conducted with Zebra fish (Daniorerio) over a96-hourexposure period.

Based on the results of the preliminary test, a saturated solution with a loading amount of 100 mg/L was prepared to obtain diluted test solutions. In a first definitive test, fish were exposed to 5 concentration levels with a spacing factor of 1.26. The concentration levels were expressed as percentage of the saturated solution, i.e. 40%SS, 50.3%SS, 63.2%SS, 79.5%SS and 100%SS. The actual concentrations of the test solutions were measured by GC/MS/MS method. A concurrent blank control was performed in parallel. Seven fish were used for the test groups and the control group.After a 96-hour exposure period, all fish were dead in all 5 test groups, therefore, a second definitive test was carried out with 4 additional lower concentrations.

In the second definitive test, according to the result of the first definitive test, the average initial concentration of 40%SS was 4.259 mg/L, the definitive test was carried out with 4 additional lower concentrations, i.e. 1.69, 2.13, 2.68, 3.38 mg/L with a spacing factor of 1.26. After the saturated solution was obtained, the actual concentration of the saturated solution was measured by GC/MS/MS method. According to the determined concentration of the saturated solution, the test solutions were diluted from the saturated solution with test water. A concurrent control group was performed in parallel.

The test was performed with a semi-static system and the test solutions were renewed every 48 hours. Dissolved oxygen and pH were measured daily during the 96-hour exposure period in the control and test solutions. Temperature was monitored continuously by an automatic temperature monitoring system and recorded at 1 hour interval.

The concentrations of test item were determined in samples of test media by GC/MS/MS method. The determined concentrations in old solution were not maintained in the range of 80% ~ 120% of initial determined concentrations, the mean exposure concentration over the whole period was calculated from the geometric mean concentration of each renewal period. For the nominal concentrations of 1.69, 2.13, 2.68, 3.38 mg/L and 40%SS, 50.3%SS, 63.2%SS, 79.5%SS and 100%SS, the mean exposure concentrations were 1.059, 1.194, 1.546, 1.948, 2.175, 2.539, 3.152, 5.255 and 7.795 mg/L.

During the 96-hour exposure period, no death and visible toxic signs were observed in the control group and the measured concentrations of 1.059, 1.194 mg/L, the fish exposed to the measured concentrations of 1.546, 1.948 mg/L showed mortality and toxicity signs such as discoloration and erratic swimming, 100% mortality occurred in the measured concentrations of 2.175, 2.539, 3.152, 5.255 and 7.795 mg/L.

According to the results and based onthe geometric mean concentration,the96-hoursmedian lethal concentration (LC₅₀) (with 95% confidence limits)of the test itemtoZebra fishwas calculated by theTrimmed Spearman-Karber (TSK) method (EPA, VERSION 1.5) as follows:

Test duration [hours]	LC50 (mg/L)	95 % lower confidence limit (mg/L)	95 % upper confidence limit (mg/L)
96	1.80	1.65	1.97
LC0(mg/L)= Highest test item concentration with 0 % mortality after 96 hours	1.194
LC100(mg/L)= Lowest test item concentration with 100 % mortality after 96 hours	2.175

 

 

Description of key information

The acute toxicity of the test substance to Danio rerio was determined in an experimental study performed in accordance with the OECD 203 test guideline and GLP requirements.

The 96h-LC50 was equal to 1.80 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.8 mg/L