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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

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 both range-finding test and 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 solution was stirred at 20°C with the glass blade at a speed avoiding formation of an emulsion. A speed of approximately 200 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 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 organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST SPECIES
The test organism used for the study was Pseudokirchneriella subcapitata, Strain No. CCAP 278/4, supplied by the Culture Centre of Algae and Protozoa (Ambleside, UK).

Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline.

The quality of the stock culture was checked for the absence of micro-organisms and deformed or abnormal cells under microscopic observation before use.

Four days before the start of the exposure, two pre-cultures were prepared by inoculating sufficient cells from the algal stock culture to the growth medium to obtain a low cell density of 104 cells/mL for pre-culturing, in order to maintain exponential growth until the start of the test. The pre-cultures were incubated under the same conditions as those used for the test cultures. Only one of the two pre-cultures was used to inoculate the test flasks for the study. The second one was only to be used if the first one was damaged.

At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to obtain an initial cell concentration of 5.103 cells/mL as recommended in the OECD 201 guideline.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 23 ± 1°C:

¿ Range-finding test: range from 22.0 to 22.5°C (mean: 22.2°C)
¿ Definitive test: range from 22.0 to 22.5°C (mean: 22.2°C)

Dissolved oxygen:

8.3 - 8.5 mg/L (range-finding test)
8.7 - 9.0 mg/L (definitive test)

Nominal and measured concentrations:

Nominal concentrations: 0 (Control), 2.4, 3.8, 6.0, 9.6, 15.6, 25.0, 40.0 (% 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:

72 h

Dose descriptor:

EC10

Effect conc.:

0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

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	< DL	< DL	< DL	< DL	NA	NA
10.0	2.29	1.74	1.45	1.83	0.428	23.4
25.0	2.64	3.37	3.59	3.20	0.499	15.6
50.0	6.56	7.66	7.24	7.16	0.555	7.8
100.0	13.16	8.88	10.29	10.78	2.178	20.2

< DL (0.033 mg/L): concentration lower than the Detection Limit

< QL (0.109 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

  

Table2Test item determination at 72h (Range-finding test)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< DL	< DL	< DL	< DL	NA	NA
10.0	0.32	0.33	0.40	0.35	0.041	11.7
25.0	0.90	0.79	0.83	0.84	0.056	6.7
50.0	2.60	2.94	2.76	2.77	0.174	6.3
100.0	4.22	3.55	3.12	3.63	0.553	15.2

< DL (0.033 mg/L): concentration lower than the Detection Limit

< QL (0.109 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

 

Table3Measured test item concentrations evolution (Range-finding test)

Test solution (% saturated stock solution)	Test item concentration(mg/L)		Deviation of 72h measured conc. from 0h (%)
	T0	T72h
Control	< DL	< DL	NA
10.0	1.83	0.35	-80.9
25.0	3.2	0.84	-73.8
50.0	7.16	2.77	-61.3
100.0	10.78	3.63	-66.3

< DL (0.033 mg/L): concentration lower than the Detection Limit

< QL (0.109 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

                                       Table4Test item determination at T0 (Definitive test)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< DL	< DL	< DL	< DL	NA	NA
2.4	0.20	0.21	0.22	0.21	0.012	5.7
3.8	0.29	0.30	0.30	0.29	0.006	2.1
6.0	0.48	0.53	0.47	0.50	0.033	6.6
9.6	0.74	0.73	0.70	0.73	0.023	3.1
15.6	1.20	1.23	1.21	1.21	0.018	1.5
25.0	1.59	1.98	1.90	1.83	0.207	11.3
40.0	2.92	2.85	2.80	2.86	0.060	2.1

< DL (0.010 mg/L): concentration lower than the Detection Limit

< QL (0.033 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

Table5Test item determination at 72h (Definitive test)

Test solution (% saturated stock solution)	Test item concentration(mg/L)			Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< DL	< DL	< DL	< DL	NA	NA
2.4	0.04	-	0.04	0.04	0.00003	0.1
3.8	0.07	0.07	0.06	0.06	0.003	4.3
6.0	0.10	0.10	0.10	0.10	0.004	3.9
9.6	0.16	0.18	0.17	0.17	0.009	5.2
15.6	0.29	0.34	0.33	0.32	0.026	8.2
25.0	0.60	0.68	0.62	0.63	0.041	6.5
40.0	1.03	0.92	1.22	1.06	0.154	14.6

< DL (0.010 mg/L): concentration lower than the Detection Limit

< QL (0.033 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

- not analysed (analytical device deficiency)

 

Table6Test item determination – Concentrations used for statistical analysis (Definitive test)

Test solution (% saturated stock solution)	Test item concentration(mg/L)		Deviation of 72h measured conc. from 0h (%)	Mean measured conc. (mg/L)*
	T0	T72h
Control	< DL	< DL	NA	NA
2.4	0.21	0.04	-81.0	0.09
3.8	0.29	0.06	-79.3	0.13
6.0	0.50	0.10	-80.0	0.22
9.6	0.73	0.17	-76.7	0.35
15.6	1.21	0.32	-73.6	0.62
25.0	1.83	0.63	-65.6	1.07
40.0	2.86	1.06	-62.9	1.74

< DL (0.010 mg/L): concentration lower than the Detection Limit

< QL (0.033 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

* Concentrations extrapolated as geometric mean of measured concentrations

Table7Algal cell density (Range-finding test)

Test solution	Replicate	Algal density at 24h (Fd, ¢ *10^4/ ml )			Algal density at 48h (Fd, ¢ *10^4/ ml )			Algal density at 72h (Fd, ¢ *10^4/ ml )			Average specific growth rate J0-J3				Specific growth rate inhibition (%)
		Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Average	Sx	CV
Control	a	2,46	2,56	10,20	11,09	10,58	7,58	61,36	58,33	6,71	1,603	1,586	0,023	1,443	Inhibition (per replicate)	Inhibition (mean)
	b	2,76			11,53			62,82			1,611
	c	2,94			11,21			57,45			1,581
	d	2,64			9,52			51,70			1,546
	e	2,33			10,03			57,24			1,580
	f	2,26			10,10			59,44			1,593
10,00	a	1,04	0,99	5,40	4,09	3,68	11,22	22,42	19,33	16,28	1,268	1,215	0,055	4,531	20,1	23,4
	b	0,94			3,27			16,13			1,158				27,0
	c	0,99			3,68			19,45			1,220				23,0
25,00	a	0,92	0,91	2,39	1,63	1,55	4,61	4,36	3,86	12,53	0,722	0,680	0,042	6,134	54,5	57,1
	b	0,92			1,52			3,40			0,639				59,7
	c	0,88			1,50			3,82			0,678				57,3
50,00	a	0,82	0,84	2,24	0,72	0,65	9,44	0,13	0,12	7,80	-0,443	-0,464	0,026	-5,705	127,9	129,3
	b	0,85			0,63			0,13			-0,456				128,7
	c	0,86			0,60			0,11			-0,494				131,1
100,00	a	0,73	0,73	0,94	0,15	0,15	13,88	0,04	0,04	12,79	-0,840	-0,811	0,041	-5,104	153,0	151,1
	b	0,72			0,14			0,04			-0,829				152,3
	c	0,74			0,18			0,05			-0,764				148,2

Table8Algal cell density (Definitive test)

Test solution	Replicate	Algal density at 24h (Fd, ¢ *10^4/ ml )			Algal density at 48h (Fd, ¢ *10^4/ ml )			Algal density at 72h (Fd, ¢ *10^4/ ml )			Average specific growth rate J0-J3				Specific growth rate inhibition (%)
		Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Average	Sx	CV
Control	a	1,88	1,67	13,16	8,04	7,04	13,19	36,78	37,22	12,41	1,433	1,435	0,039	2,745	Inhibition (per replicate)	Inhibition (mean)
	b	1,97			8,09			37,85			1,442
	c	1,71			7,17			36,61			1,431
	d	1,41			5,74			32,94			1,396
	e	1,54			6,36			33,40			1,401
	f	1,52			6,87			45,76			1,506
2,40	a	1,33	1,31	2,29	6,85	6,49	4,94	31,02	29,25	7,18	1,376	1,356	0,024	1,790	4,1	5,5
	b	1,28			6,23			26,93			1,329				7,4
	c	1,33			6,39			29,80			1,363				5,0
3,80	a	1,32	1,27	4,80	6,43	6,41	0,39	33,91	31,95	5,30	1,406	1,385	0,017	1,258	2,0	3,4
	b	1,20			6,38			30,88			1,374				4,2
	c	1,27			6,43			31,08			1,377				4,1
6,00	a	1,27	1,09	14,42	5,90	5,58	5,36	31,06	29,82	4,90	1,376	1,362	0,016	1,211	4,1	5,0
	b	0,98			5,31			30,19			1,367				4,7
	c	1,02			5,53			28,21			1,344				6,3
9,60	a	0,81	0,94	12,18	4,52	4,64	2,31	22,73	23,71	10,66	1,272	1,285	0,035	2,703	11,3	10,4
	b	1,04			4,72			21,82			1,259				12,3
	c	0,95			4,69			26,59			1,325				7,7
15,60	a	0,78	0,76	5,64	3,30	3,10	5,48	16,18	15,89	3,11	1,159	1,153	0,010	0,909	19,2	19,7
	b	0,78			3,00			16,16			1,159				19,2
	c	0,71			3,01			15,31			1,141				20,5
25,00	a	0,65	0,66	3,12	1,56	1,65	7,01	5,54	6,09	13,21	0,802	0,832	0,043	5,136	44,1	42,0
	b	0,64			1,78			7,02			0,880				38,6
	c	0,68			1,61			5,73			0,813				43,4
40,00	a	0,63	0,63	0,42	0,86	0,85	2,48	1,02	1,15	10,60	0,237	0,277	0,036	13,040	83,5	80,7
	b	0,64			0,87			1,19			0,288				79,9
	c	0,63			0,83			1,26			0,307				78,6

Table9Percentage of inhibition (Range-finding test)

Test solution (% saturated stock solution)	Growth rate Iµi (%)	YieDL Iyi (%)
10.0	23.4	67.4
25.0	57.1	94.2
50.0	>100	>100
100.0	>100	>100

Iµi: average percentage inhibition of growth rate

Iyi: average percentage inhibition of yieDL

  

Table10Percentage of inhibition (Definitive test)

Test solution (% saturated stock solution)	Growth rate Iµi (%)	YieDL Iyi (%)
2.4	5.5	21.7
3.8	3.4	14.4
6.0	5.0	20.2
9.6	10.4	36.8
15.6	19.7	58.1
25.0	42.0	84.8
40.0	80.7	98.2

Iµi: average percentage inhibition of growth rate

Iyi: average percentage inhibition of yieDL

Table11Measured pH and dissolved oxygen (Range-finding test)

Test solution (% saturated stock solution)	pH		Dissolved O2 (mg/L)
	0h	72h	0h	72h
Control	7.9	7.9	8.4	8.5
10.0	8.0	7.9	8.3	8.4
25.0	8.0	7.9	8.3	8.4
50.0	8.0	7.9	8.3	8.3
100.0	7.9	7.9	8.4	8.3

  

Table12Measured pH and dissolved oxygen (Definitive test)

Test solution (% saturated stock solution)	pH		Dissolved O2 (mg/L)
	0h	72h	0h	72h
Control	7.9	8.0	9.0	8.8
2.4	7.9	8.0	9.0	8.8
3.8	7.9	8.0	9.0	8.8
6.0	7.9	8.0	9.0	8.8
9.6	7.9	8.0	9.0	8.8
15.6	7.9	7.9	9.0	8.8
25.0	7.9	7.9	9.0	8.7
40.0	7.9	7.9	9.0	8.7

NA: Not Applicable

 

Reference item test

The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate. The latest value of ErC50 obtained in April 2018 was 1.15 mg/L.

 

For information, according to ISO 8692 standard, 72h-ErC50 obtained from an inter-laboratory exercise on potassium dichromate was in the 0.65 to 1.73 mg/L range.

Range-finding test

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

 

Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape ofP. subcapitataalgae is a crescent shaped cell with an average length of 5-10 µm.

The analytical results data are presented inTable1toTable3. Chemical analysis of test samples taken at 0 and 72 h indicated that concentrations of the test item were not maintained over the 72h test period (i.e. deviation > 20%). The instability of the substance was thus confirmed over the test period.

 

The examination of growth plots shows exponential growth of control culture at the expected rate throughout the test.

 

Cell densities, biomass, and growth rate are summarised inTable7, percentages of inhibition inTable9.The results (growth rate) demonstrated that algal growth was significantly inhibited from the lowest tested concentration.

  

Definitive test

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

 

Microscopic observation confirmed that the algae appeared "normal" at the end of the test: the normal shape ofP. subcapitataalgae is a crescent shaped cell with an average length of 5-10 µm.

 

The analytical results data are presented in       Table 4toTable6. Chemical analysis of test samples taken at 0 and 72 h indicated that concentrations of the test item were not maintained over the 72h test period (i.e. deviation > 20%). As a consequence, the stability of the substance was not confirmed over the test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.

 

The examination of growth plots shows exponential growth of control culture at the expected rate throughout the test.

 

Cell densities, biomass, and growth rate throughout the test period are summarised inTable8. The percentages of inhibition are presented inTable10.The results (growth rate) demonstrated that algal growth was inhibited from 3.4% to 80.7% at the highest tested concentration.

Environmental conditions

Dissolved oxygen and pH are presented inTable11&Table12. Measurements were carried out in non-inoculated solution at T₀and inoculated solution at T₇₂.

The light intensity was measured as follows:

 

§ Range-finding test: range from 108 to 110 µE.m².s^-1

§ Definitive test: range from 108 to 119 µE.m².s^-1

Statistical analysis

Statistical analysis was performed with the software ToxRat^®version 3.2.1.

Validation criteria

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

 

§ The biomass in the control cultures increased exponentially by a factor of 74.5 within the 72-hour test period (required: at last 16-fold)

 

§ The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were less than or equal to 16.3 (required: < 35%)

 

§ The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 2.7% (required: < 7%)

Validity criteria fulfilled:

yes

Conclusions:

DISCUSSION/CONCLUSION
The results are reported as growth rate and yield inhibitions. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined.

Chemical analysis of the test item showed the instability of the substance over the 72-hour test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.

Growth rate:
72-h ErC10 = 0.55 mg/L (95% confidence level: 0.48 – 0.63 mg/L)
72-h ErC50 = 1.15 mg/L (95% confidence level: 0.98 – 1.35 mg/L)

Yield:
72-h EyC10 = 0.16mg/L (95% confidence level: 0.11 – 0.25 mg/L)
72-h EyC50 = 0.50 mg/L (95% confidence level: 0.31 – 0.83 mg/L)

Executive summary:

This study was designed to determine the effects of the test item on the growth ofPseudokirchneriella subcapitatain a 72-hour test according to the OECD 201 Guideline.

 

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

 

Chemical analysis of the test item showed the instability of the substance over the 72-hour test period. Based on these results, the exposure concentrations were based on the geometric mean of measured concentrations.

 

The results are reported as growth rate and yield inhibitions. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) and yield (72h-EyC10 and 72h-EyC50, respectively) were determined.

Growth Function	72h-EC10(mg/L)	95% confidence level		72h-EC50 (mg/L)	95% confidence level
		Lower limit	Upper limit		Lower limit	Upper limit
Growth rate	0.55	0.48	0.63	1.15	0.98	1.35
Yield	0.16	0.11	0.25	0.50	0.31	0.83

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

 

§ The biomass in the control cultures increased exponentially by a factor of 74.5 within the 72-hour test period (required: at last 16-fold)

 

§ The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were less than or equal to 16.3 (required: < 35%)

 

§ The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 2.7% (required: < 7%)

 

Description of key information

The toxicity of the test substance to Pseudokirchneriella subcapitata was determined in an experimental study performed in accordance with the OECD 201 test guideline and GLP requirements.

The 72h-ErC50 was equal to 1.15 mg/L and the 72h-ErC10 was equal to 0.55 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.15 mg/L

EC10 or NOEC for freshwater algae:

0.55 mg/L