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

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:

2018-04-19 to 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)

Version / remarks:

One deviation was observed in this study: one series of analyses was omitted during the definitive test #1 (fresh solutions prepared at 24h), which could have led to an error when calculating exposure concentrations.

A second definitive test was thus conducted with identical test conditions to confirm the test results. Both test results are nearly identical, this deviation was thus considered to have not affected the outcome or the achievement of the study objectives.

Deviations:

yes

Remarks:

See "Version / remarks".

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

In the absence of analytical method described and provided by the Sponsor, a method has been developed and validated according to the protocol as specified in the Standard Operating Procedure "MSG-MO-001 Traitement d'une demande d'analyse en BPL".

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS
For the range-finding test and both definitive tests, as the test item was known to be difficult to dissolve, a saturated solution was prepared using slow-stirring conditions described in the OECD 123 guideline.

The method used a special temperature controlled glass-jacketed test vessel (around 1000 mL of capacity) with a glass blade at its top. A known volume of test item (1000 µL) was first poured into the flask, then a known quantity of test medium (1000 mL which corresponds to a loading rate of 1 g/L) was gently added. The solutions was stirred at 20°C with the glass blade at a speed avoiding formation of an emulsion. A speed of approximately 200/260 rpm was set up in order to create a vortex depth of around 0.5 cm: the aqueous phase was kept under stirring during approximately 24 to 26 hours, and then drawn off as a clear solution after a rest phase of a few minutes without agitation. Tests solutions were then prepared from dilutions of this saturated solution.

The pH of the test solutions were not adjusted.

Test solutions were only renewed during the definitive test, at 24h from a test item stock solution prepared freshly (semi-static test).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SPECIES
The test organisms used for this study were Daphnia magna Straus (Cladocera, Crustacea), clone 5, bred within the laboratory by acyclical parthenogenesis in a synthetic medium. Daphnia magna cultures were fed with a mixture of the microalgae Chlorella vulgaris and Pseudokirchneriella subcapitata. The neonates used were less than 24h old at test initiation (selected by filtration) and were not first brood progeny.

Test animals were not fed during the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

The total hardness was measured at 250 mg CaCO3/L in the control group at the test initiation for both range-finding and definitive test #1 and at 230 mg CaCO3/L for the definitive test #2.

Test temperature:

Temperatures were maintained at 20 ± 1°C:

• Preliminary test: range from 20.3 to 20.4°C
• Definitive test #1: range from 20.2 to 20.4°C
• Definitive test #2: range from 20.2 to 20.5°C

pH:

• Preliminary test: range from 7.8 to 8.0
• Definitive test #1: range from 7.9 to 8.0
• Definitive test #2: range from 7.9 to 8.1

Dissolved oxygen:

• Preliminary test: range from 8.0 to 8.5 mg/L
• Definitive test #1: range from 8.0 to 8.6 mg/L
• Definitive test #2: range from 7.1 to 8.4 mg/L

Nominal and measured concentrations:

Nominal concentrations:
• Preliminary test: 0 (Control), 10, 25, 50, 100% saturated stock solution
• Definitive test #1: 0 (Control), 9.55, 15.25, 24.40, 39.05, 62.50, 100% saturated stock solution
• Definitive test #2: 0 (Control), 9.55, 15.25, 24.40, 39.05, 62.50, 100% saturated stock solution

Measured concentrations: see "Any other information on results incl. tables".

Details on test conditions:

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

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

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

Results and discussion

Table1Test item determination at T0 (Range-finding test)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	< LD	NA	NA
10.0	0.85	0.98	0.80	0.88	0.09	10.8
25.0	2.04	2.38	2.65	2.35	0.30	12.9
50.0	4.65	4.61	4.82	4.69	0.11	2.4
100.0	7.49	8.03	7.94	7.82	0.29	3.7

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

  

Table2Test item determination at 48h (Range-finding test)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	< LD	NA	NA
10.0	0.54	0.46	0.51	0.50	0.04	7.8
25.0	1.10	1.16	0.76	1.01	0.21	21.3*
50.0	2.80	3.05	2.57	2.81	0.24	8.5
100.0	4.75	5.47	5.19	5.14	0.36	7.0

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

* %RSD > 20%. the result is provided for information only

  

Table3Test item determination – Concentrations used for statistical analysis (Range-finding test)

Test solution (% saturated stock solution)	Test item concentration(mg/L)		Deviation of 48h measured conc. from 0h (%)
	T0	T48h
Control	< LD	< LD	NA
10.0	0.88	0.5	-43.2
25.0	2.35	NA	NA
50.0	4.69	2.81	-40.1
100.0	7.82	5.14	-34.3

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

 

Table4Test item determination at T0 (Definitive test #1. fresh solutions)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	< LD	NA	NA
9.55	0.69	0.70	0.71	0.70	0.010	1.4
15.25	1.37	1.32	1.40	1.36	0.039	2.9
24.40	2.37	1.73	1.98	2.03	0.320	15.8
39.05	3.21	2.69	3.29	3.06	0.327	10.7
62.50	5.15	4.83	4.62	4.87	0.268	5.5
100.00	6.64	6.47	4.89	6.00	0.965	16.1

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

Table5Test item determination at T24h (Definitive test #1. old solutions)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	< LD	NA	NA
9.55	0.50	0.55	0.60	0.55	0.051	9.2
15.25	0.88	0.85	0.86	0.87	0.015	1.7
24.40	1.30	1.73	1.69	1.57	0.242	15.4
39.05	2.04	2.41	2.04	2.16	0.213	9.8
62.50	2.90	3.31	3.60	3.27	0.355	10.9
100.00	4.88	3.88	4.21	4.32	0.510	11.8

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

  

Table6Test item determination at 48h (Definitive test #1. old solutions)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	< LD	NA	NA
9.55	0.40	0.39	0.40	0.40	0.007	1.7
15.25	0.68	0.65	0.84	0.72	0.105	14.6
24.40	1.04	1.22	0.94	1.07	0.143	13.4
39.05	1.54	1.43	1.70	1.55	0.136	8.8
62.50	2.74	2.48	2.44	2.55	0.163	6.4
100.00	4.90	3.20	3.01	3.70	1.042	28.1*

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

* %RSD > 20%: reliability of this data is low, but not disregarded for the final calculation

  

Table7Test item determination – Concentrations used for statistical analysis (Definitive test #1)

Test solution (% saturated stock solution)	Test item concentrations(mg/L)						Exposure conc. (mg/L)*
	T0/T24			T24/T48
	fresh	old	Deviation T0/T24	fresh	old	Deviation T24/T48
Control	<DL	<DL	NA	<DL	<DL	NA	NA
9.55	0.7	0.55	-21.4	ND	0.4	NA	0.54
15.25	1.36	0.87	-36.0	ND	0.72	NA	0.95
24.40	2.03	1.57	-22.7	ND	1.07	NA	1.51
39.05	3.06	2.16	-29.4	ND	1.55	NA	2.17
62.50	4.87	3.27	-32.9	ND	2.55	NA	3.44
100.00	6	4.32	-28.0	ND	3.7	NA	4.58

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

ND: Not Determined. see paragraph1.6.

*Concentrations extrapolated as geometric mean of measured concentrations

Table8Test item determination at T0 (Definitive test #2. fresh solutions)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	< LD	NA	NA
9.55	0.49	0.38	0.55	0.47	0.09	18
15.25	1.05	0.75	0.87	0.89	0.15	17
24.40	1.56	1.21	1.29	1.35	0.19	14
39.05	2.54	1.83	1.69	2.02	0.45	22*
62.50	3.86	2.73	3.11	3.23	0.58	18
100.00	5.99	4.34	4.77	5.04	0.86	17

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

* %RSD > 20%: reliability of this data is low, but not disregarded for the final calculation

  

Table9Test item determination at T24h (Definitive test #2. old solutions)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	< LD	NA	NA
9.55	0.33	0.41	0.36	0.37	0.04	9.6
15.25	0.56	0.57	0.72	0.62	0.09	14
24.40	1.02	0.91	1.17	1.04	0.13	13
39.05	1.50	1.49	1.76	1.58	0.15	9.5
62.50	2.05	2.39	2.59	2.34	0.28	12
100.00	3.35	3.91	4.17	3.81	0.42	11

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

Table10Test item determination at 48h (Definitive test #2. Fresh solutions)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	< LD	NA	NA
9.55	0.63	0.60	0.63	0.62	0.02	2.6
15.25	0.92	0.86	1.00	0.92	0.07	7.6
24.40	1.52	1.34	1.47	1.44	0.09	6.4
39.05	2.72	2.44	2.40	2.52	0.18	7.0
62.50	3.94	3.59	4.91	4.15	0.68	16
100.00	5.54	5.35	5.71	5.54	0.18	3.2

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

  

Table11Test item determination at 48h (Definitive test #2. old solutions)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	< LD	NA	NA
9.55	0.33	0.31	0.35	0.33	0.02	4.9
15.25	0.49	0.50	0.52	0.50	0.02	3.3
24.40	1.06	0.95	1.05	1.02	0.06	6.2
39.05	1.48	1.30	1.61	1.47	0.16	11
62.50	1.98	2.26	2.37	2.20	0.20	9.2
100.00	3.58	3.81	3.95	3.78	0.19	5.0

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

Table12Test item determination – Concentrations used for statistical analysis (Definitive test #2)

Test solution (% saturated stock solution)	Test item concentrations(mg/L)						Exposure conc. (mg/L)*
	T0/T24			T24/T48
	fresh	old	Deviation T0/T24	fresh	old	Deviation T24/T48
Control	<DL	<DL	NA	<DL	<DL	NA	NA
9.55	0.47	0.37	-22.6	0.62	0.33	-47.3	0.43
15.25	0.89	0.62	-31.0	0.92	0.50	-45.3	0.71
24.40	1.35	1.04	-23.6	1.44	1.02	-29.2	1.20
39.05	2.02	1.58	-21.5	2.52	1.47	-41.9	1.85
62.50	3.23	2.34	-27.6	4.15	2.20	-47.0	2.88
100.00	5.04	3.81	-24.4	5.54	3.78	-31.7	4.48

< DL (0.034 mg/L): concentration lower than the Detection Limit

< QL (0.112 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

*Concentrations extrapolated as geometric mean of measured concentrations

Table13Measured pH and dissolved oxygen (Range-finding test)

Test solution (% saturated stock solution)	pH		Dissolved O2 (mg/L)
	0h	48h	0h	48h
Control	8.0	7.8	8.5	8.0
10.0	8.0	7.8	8.4	8.2
25.0	8.0	7.9	8.4	8.4
50.0	8.0	7.9	8.4	8.3
100.0	8.0	7.8	8.3	8.1

 

Table14Measured pH (Definitive test #1)

Test solution (% saturated stock solution)	T0	T24		T48h
	fresh sol.	old sol.	fresh sol.	old sol.
Control	8.0	7.9	8.0	7.9
9.55	8.0	7.9	8.0	8.0
15.25	8.0	7.9	8.0	8.0
24.40	8.0	8.0	8.0	8.0
39.05	8.0	8.0	8.0	8.0
62.50	8.0	8.0	8.0	8.0
100.00	8.0	8.0	8.0	8.0

Table15Measured dissolved oxygen (Definitive test #1)

Test solution (% saturated stock solution)	T0	T24		T48h
	fresh sol.	old sol.	fresh sol.	old sol.
Control	8.4	8.0	8.7	8.2
9.55	8.6	8.1	8.6	8.2
15.25	8.6	8.1	8.6	8.2
24.40	8.6	8.1	8.6	8.1
39.05	8.6	8.1	8.6	8.2
62.50	8.5	8.2	8.6	8.2
100.00	8.4	8.2	8.5	8.2

Table16Measured pH (Definitive test #2)

Test solution (% saturated stock solution)	T0	T24		T48h
	fresh sol.	old sol.	fresh sol.	old sol.
Control	8.0	7.9	8.0	7.9
9.55	8.1	8.0	8.0	8.0
15.25	8.1	8.0	8.0	8.0
24.40	8.0	8.0	8.0	8.0
39.05	8.0	8.0	8.0	8.0
62.50	8.0	8.0	8.0	8.0
100.00	8.0	8.0	8.0	8.0

Table17Measured dissolved oxygen (Definitive test #2)

Test solution (% saturated stock solution)	T0	T24		T48h
	fresh sol.	old sol.	fresh sol.	old sol.
Control	8.4	6.8	7.8	7.7
9.55	8.0	7.5	7.7	7.7
15.25	8.1	7.6	7.4	7.4
24.40	8.1	7.5	7.1	7.7
39.05	7.9	7.6	7.8	7.7
62.50	8.1	7.6	7.7	7.8
100.00	8.1	7.5	7.8	7.8

  Table18Cumulative immobilisation (Range-finding test)

Test solution (% saturated stock solution)	Total daphnids at T0	Immobile daphnids		Mobility inhibition at 48h(%)
		24h	48h
Control	20	0	0	0.0%
10.0	20	0	2	10.0%
25.0	20	1	1	5.0%
50.0	20	2	7	35.0%
100.0	20	20	20	100.0%

 

Table19Cumulative immobilisation (définitive test #1)

Test solution (mg/L)	Total daphnids at T0	Immobile daphnids		Mobility inhibition at 48h(%)
		24h	48h
Control	20	0	0	0.0%
9.55	20	0	1	5.0%
15.25	20	0	0	0.0%
24.40	20	0	0	0.0%
39.05	20	2	12	60.0%
62.50	20	11	20	100.0%
100.00	20	15	19	95.0%

  

Table20Cumulative immobilisation (Définitive test #2)

Test solution (mg/L)	Total daphnids at T0	Immobile daphnids		Mobility inhibition at 48h(%)
		24h	48h
Control	20	0	0	0.0%
9.55	20	0	0	0.0%
15.25	20	0	0	0.0%
24.40	20	1	5	25.0%
39.05	20	1	9	45.0%
62.50	20	0	12	60.0%
100.00	20	0	12	60.0%

Reference item test

The sensitivity of the test system and the methodology areevaluated monthlyby performing an acuteDaphnia magnatoxicity test on potassium dichromate. The most recent values of 24h-EC50, obtained in April 2018 and July 2018, are as follows:

 

24h-EC50 = 1.06 mg/L (April 2018)

24h-EC50 = 0.8 mg/L (July 2018)

 

The ISO 6341 norm has a validity criterion specifying that the reference item EC50 must be in the 0.6 to 2.1 mg/L range.

Range-finding test

The numbers of immobileD. magnarecorded over the test period are presented inTable18.

 

Test item concentration results are presented inTable1toTable3. Chemical analysis of test samples taken at 0 and 48h indicated that measured concentrations of the test item were not maintained over the 48h test period (i.e. deviation >20%). As a consequence, the stability of the substance was not confirmed over the test period; it was decided to perform the definitive test under semi-static conditions (daily renewal).

 

Definitive tests #1 & #2

The numbers of immobileD. magnarecorded over the test period are presented inTable19(Definitive test #1) and inTable20(Definitive test #2).

 

Test item concentration results are presented inTable4toTable7for the definitive test #1 and inTable8toTable12for the definitive test #2. Chemical analysis of the test item showed the instability of the substance over both 48-hours tests period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.

Environmental conditions

The appearance of the test solutions was visually checked at the beginning and at the end of the tests. Solutions were found to be clear, no precipitation was observed at the end of the test.

 

Dissolved oxygen and pH measurements are presented inTable13toTable16.

 

The light intensity was measured as follows:

·        Preliminary test: range from 962 to 986 lux

·        Definitive test #1: range from 970 to 1148 lux

·        Definitive test #2: range from 780 to 881 lux

Statistical analysis

Statistical analysis was performed with the software ToxRatPro Version 3.2.1. A Probit analysis using linear max. likelihood regression was statistically considered to be more adapted to the raw data obtained.

  

Validation criteria

The definitive tests met the validity criteria of the test guideline detailed as follows:

 

§ Immobilisation in the control did not exceed 10% at the end of both tests (0%)

§ Dissolved oxygen concentrations remained above 3 mg/L over both test periods.

Validity criteria fulfilled:

yes

Conclusions:

DISCUSSION/CONCLUSION
The geometric means of measured concentrations were used to determine 48h-EC10 and 48h-EC50:

Definitive test #1 (semi-static test)
48h-EC10 = 1.24 mg/L (95% confidence level: not determined due to mathematical reasons or inappropriate data)
48h-EC50 = 2.09 mg/L (95% confidence level: not determined due to mathematical reasons or inappropriate data)

Definitive test #2 (semi-static test)
48h-EC10 = 0.87 mg/L (95% confidence level: 0.50 – 1.17 mg/L)
48h-EC50 = 2.64 mg/L (95% confidence level: 2.09 – 3.66 mg/L)

Although one series of analyses was omitted during the definitive test #1, results are nearly identical. Nevertheless, the results of definitive test #2 are considered to be a more reliable estimation of effective concentrations (48h-EC10 = 0.87 mg/L, 48h-EC = 2.64 mg/L), as all analytical measurements were properly performed and taken into account when calculating geometric means.

Executive summary:

Summary

This study was designed to determine the effects of the test item toDaphnia magnaover a 48h period with semi-static conditions, according to the OECD 202 Guideline.

 

Note: by mistake, one series of analyses was not carried out during the definitive test leading to the performance of a second definitive test carried out with identical conditions. Both test results are reported here as "Definitive test #1" and "Definitive test #2".

 

As the test item was known to be difficult to dissolve in water, tests solutions were prepared from dilutions of a saturated solutionprepared using slow-stirring conditionsdescribed in the OECD 123 guideline.

 

As there was evidence that the concentrations of the substance being tested were not satisfactorily maintained within ± 20% of the measured initial concentration throughout both definitive tests, the exposure concentrations were based on the geometric mean of measured concentrations:

 

			95% confidence level
			Lower limit	Upper limit
Definitive test #1 (semi-static test)	48h-EC10	1.24 mg/L	ND	ND
	48h-EC50	2.09 mg/L	ND	ND
Definitive test #2 (semi-static test)	48h-EC10	0.87 mg/L	0.50	1.17
	48h-EC50	2.64 mg/L	2.09	3.66

 

Although one series of analyses was omitted during the definitive test #1, results are nearly identical.

Nevertheless, the results of definitive test #2 are considered to be a more reliable estimation of effective concentrations (48h-EC10 = 0.87 mg/L, 48h-EC = 2.64 mg/L, as all analytical measurements were properly performed and taken into account when calculating geometric means.

The definitive tests met the validity criteria of the test guideline detailed as follows:

 

§ Immobilisation in the control did not exceed 10% at the end of both tests (0%)

§ Dissolved oxygen concentrations remained above 3 mg/L over both test periods.

Description of key information

The acute toxicity of the test substance to Daphnia magna was determined in an experimental study performed in accordance with the OECD 202 test guideline and GLP requirements.

Following a preliminary range-finder test, a definitive test was carried out. In this test, one series of test solution analyses was not carried out by error, which led to the performance of a second definitive test carried out with identical conditions. This second definitive test resulted in a reliable estimation of effective concentrations, as all analytical measurements were properly performed and taken into account when calculating geometric means.

According to the results of the second definitive test, the 48h-EC50 was equal to 2.64 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.64 mg/L