3,3,5-trimethylcyclohexyl 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:

2016-09-15 to 2017-

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:

Samples were taken from all test concentrations and the control at the start of the test and after 72 hours of exposure. Inoculated samples were previously filtered through 0.45µm then analysed according to the analytical procedure.

Vehicle:

no

Details on test solutions:

As the test item was suspected to be unstable in water (due to its chemical nature), 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 sampling tap at the lower end and a magnetic stir bar at its bottom. A known volume of test medium (around 1000 mL) was first poured into the flask, then a known quantity of test item (107.5 µL) was gently added at the surface of medium (loading rate of 100 mg/L). The solution was stirred at room temperature with a magnetic stir bar at a speed avoiding formation of an emulsion. The speed 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 hours and then drawn off as a clear solution after a rest phase of 30 min. without agitation. Tests solutions were then prepared from dilutions of this saturated solution.

Test organisms (species):

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

Details on test organisms:

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 into the growth medium to reach a low cell density, e.g. 2.103 cells/mL to 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 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

Hardness:

Dilution water is prepared according to the protocol described in Annex 3 of the OECD guideline 201.

Test temperature:

Temperature was maintained at 23 ± 1°C: range from 22.3 to 23.2°C (mean: 22.7°C)

pH:

7.9 - 8.1

Dissolved oxygen:

9.1 - 9.5

Nominal and measured concentrations:

Dilutions from a saturated stock solution were prepared: 10.0, 14.8, 21.6, 31.8, 46.6, 68.2 and 100% v/v
Concentrations were determined at beginning and end of exposure period where the test item was no more detected, see table below. Results are given with respect to geometric means:

Details on test conditions:

The incubation was conducted in a phytoculture cabinet that allows test flasks to be incubated under precise conditions: temperature was set to 23 ± 1°C. Flasks were continuously shaken with a rotation at around 100 rpm and constantly illuminated by fluorescent tubes between 6,000 and 10,000 lux.

Glass bottles (120 mL capacity) capped with cellulose bungs were used as test vessels. They were filled with a volume of 50 mL.

Algae were exposed to a series of dilutions (10.0, 14.8, 21.6, 31.8, 46.6, 68.2 and 100% v/v) of the saturated stock solution in dilution water. An inoculated control flask was prepared and incubated under the same conditions with no test item. Three vessels were prepared at each test concentration and six vessels for the control group. The test was started using an initial cell concentration of ca 0.5 x 104 cells/mL.

Algal cell concentrations were measured in each flask at 24, 48 and 72 h using flow cytometry (Guava easyCyte™ flow cytometer Merck Millipore). Cell concentrations were determined using 96 wells single use microplates and a laser beam at 488 nm. After 24 and 48h of incubation, aliquot of 200 µL was sampled from each inoculated test flask, pipetted into a microplate. After 72 h, the samples with high cell density were diluted to ¼ (50 µL + 150 µL of filtered dilution water) and pipetted into a microplate. Time between sampling and measurement is approximately 15 – 30 min

The pH and dissolved oxygen concentrations were measured in all test solutions, in non-inoculated flask at the beginning of the test and in inoculated flasks at the end of the test.

The temperature in the incubator was continuously recorded throughout the test.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

not determinable

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 August 2016 was 1.57 mg/L.

 

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

 

Algae 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 of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm.

 

The analytical results data are presented in the tables below:

Test item determination at T0

Nominal concentration (% v/v saturated solution)	Test item concentration(mg/L)			Dilution	Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	2	NA	NA	NA
10.0	0.65	0.63	0.67	2	1.30	0.038	2.9
14.8	0.98	0.98	1.01	2	1.98	0.040	2.0
21.6	1.49	1.51	1.49	2	2.99	0.026	0.9
31.8	2.13	2.13	2.11	2	4.25	0.023	0.5
46.6	3.19	3.22	3.22	2	6.42	0.040	0.6
68.2	4.52	4.56	4.58	2	9.11	0.054	0.6
100.0	7.10	7.03	7.01	2	14.09	0.088	0.6

< DL (0.025 mg/L): concentration lower than the Detection Limit of the analytical method

< QL (0.080 mg/L): concentration lower than the Quantification Limit of the analytical method

NA: Not Applicable

Test item determination at 72h

Nominal concentration (% v/v saturated solution)	Test item concentration(mg/L)			Dilution	Mean	SD	RSD (%)
	Measure 1	Measure 2	Measure 3
Control	< LD	< LD	< LD	2	NA	NA	NA
10.0	< LD	< LD	< LD	2	NA	NA	NA
14.8	< LD	< LD	< LD	2	NA	NA	NA
21.6	< LD	< LD	< LD	2	NA	NA	NA
31.8	< LD	< LD	< LD	2	NA	NA	NA
46.6	< LD	< LD	< LD	2	NA	NA	NA
68.2	< LD	< LD	< LD	2	NA	NA	NA
100.0	< LD	< LD	< LD	2	NA	NA	NA

< DL (0.025 mg/L): concentration lower than the Detection Limit of the analytical method

< QL (0.080 mg/L): concentration lower than the Quantification Limit of the analytical method

NA: Not Applicable

Test item determination – Used concentrations

Nominal concentration (% v/v saturated solution)	Test item concentration(mg/L)		Deviation of 72h measured conc. from 0h (%           )	Retained conc. (mg/L)*
	T0	T72h
Control	< LD	< LD	NA	NA
10.0	1.30	< LD	>90%	0.18
14.8	1.98	< LD	>90%	0.22
21.6	2.99	< LD	>90%	0.27
31.8	4.25	< LD	>90%	0.33
46.6	6.42	< LD	>90%	0.40
68.2	9.11	< LD	>90%	0.48
100.0	14.09	< LD	>90%	0.59

< DL (0.025 mg/L): concentration lower than the Detection Limit of the analytical method

< QL (0.080mg/L): concentration lower than the Quantification Limit of the analytical method

NA: Not Applicable

*Concentrations extrapolated as geometric means of measured concentrations

Chemical analysis of test samples taken at 0 and 72h 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 and geometric means of measured concentrations were thus used to determine EC10 and EC50.

 

Cell densities, biomass and growth rate throughout the test period are summarised in the table below:

Algal cell density

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	2.37	2.16	6.67	9.38	9.27	6.20	47.33	47.90	6.33	1.517	1.520	0.021	1.392	Inhibition (per replicate)	Inhibition (mean)
	b	2.27			9.96			51.93			1.548
	c	2.02			9.38			47.41			1.517
	d	2.11			9.72			50.67			1.540
	e	2.00			8.44			43.47			1.488
	f	2.18			8.76			46.58			1.511
10.00	a	2.02	1.96	6.79	9.85	9.70	2.75	44.55	45.86	4.12	1.497	1.506	0.014	0.903	1.5	0.9
	b	1.81			9.40			45.00			1.500				1.3
	c	2.05			9.86			48.03			1.522				-0.1
14.80	a	1.84	1.71	15.73	7.90	8.49	10.89	38.73	42.12	10.79	1.450	1.477	0.035	2.379	4.6	2.9
	b	1.40			8.02			40.34			1.463				3.7
	c	1.88			9.56			47.28			1.516				0.2
21.60	a	1.61	1.60	6.68	8.72	8.20	8.40	39.35	39.34	0.41	1.455	1.455	0.001	0.094	4.3	4.3
	b	1.71			8.47			39.50			1.456				4.2
	c	1.50			7.42			39.18			1.454				4.4
31.80	a	1.60	1.26	23.43	8.39	6.76	20.95	39.25	35.19	10.31	1.454	1.417	0.034	2.381	4.3	6.8
	b	1.10			6.03			32.26			1.389				8.6
	c	1.09			5.86			34.06			1.407				7.4
46.60	a	1.03	1.16	14.18	5.88	6.08	9.41	30.54	32.92	7.90	1.371	1.395	0.026	1.875	9.8	8.2
	b	1.10			5.65			32.52			1.392				8.5
	c	1.34			6.73			35.70			1.423				6.4
65.20	a	0.97	0.96	3.24	4.82	4.49	7.34	24.94	24.88	10.14	1.303	1.301	0.034	2.612	14.3	14.4
	b	0.92			4.16			22.33			1.266				16.7
	c	0.98			4.48			27.37			1.334				12.2
100.00	a	0.68	0.70	3.74	2.94	3.05	6.13	11.88	12.34	9.00	1.056	1.068	0.029	2.751	30.5	29.8
	b	0.69			2.94			11.53			1.046				31.2
	c	0.73			3.26			13.60			1.101				27.6

The percentages of inhibition are presented in the table below:

Percentage of inhibition

Nominal concentration(% v/v saturated solution)	Geometric means measured conc. (mg/L)	Growth rate Iµi (%)	Yield Iyi (%)
10.0	0.18	0.9	4.3
14.8	0.22	2.9	12.2
21.6	0.27	4.3	18.0
31.8	0.33	6.8	26.8
46.6	0.40	8.2	31.6
68.2	0.48	14.4	48.6
100.0	0.59	29.8	75.0

Iµi: average percentage inhibition of growth rate

Iyi: average percentage inhibition of yield

The results (growth rate) demonstrated that algal growth was inhibited from 0.9% at the lowest concentration (i.e. 10.0% of dilution) to 29.8% at the highest tested concentration (i.e. saturation concentration of the test item).

Validation criteria

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

* The biomass in the control cultures increased exponentially by a factor of 95.8 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 was max 7.8% (required: < 35%) 

* The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 1.4% (required: < 7%)

Validity criteria fulfilled:

yes

Conclusions:

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

As the test item was suspected to be unstable in water (due to its chemical nature), tests solutions were prepared from dilutions of a saturated solution prepared using slow-stirring conditions described in the OECD 123 guideline.

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:
72h-EC10 = 0.43 mg/L (95% CI: 0.40 - 0.45 mg/L)
72h-EC50 = Not determined due to mathematical reasons or inappropriate data
Yield:
72h-EC10 = 0.24 mg/L (95% CI: 0.20 - 0.29 mg/L)
72h-EC50 = 0.47 mg/L (95% CI: 0.36 - 0.60 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 suspected to be unstable in water (due to its chemical nature), tests solutions were prepared from dilutions of a saturated solution prepared using slow-stirring conditions described in the OECD 123 guideline.

 

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.

 

As the analytical determination of the test item concentrations throughout the test period have shown unstability of the substance, geometric means of measured concentrations were used to determine 72h-EC10 and 72h-LC50 (all results are mg/L):

Growth Rate:

72h-EC10 = 0.43 mg/L (95% CI: 0.40 - 0.45 mg/L)

72h-EC50 = Not determined due to mathematical reasons or inappropriate data

Yield:

72h-EC10 = 0.24 mg/L (95% CI: 0.20 - 0.29 mg/L)

72h-EC50 = 0.47 mg/L (95% CI: 0.36 - 0.60 mg/L)

Thetest met the validity criteria of the test guideline detailed as follows:

 

§ The biomass in the control cultures increased exponentially by a factor of 95.8 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 was max 7.8% (required: < 35%)

 

§ The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 1.4% (required: < 7%)

Description of key information

A study assessing the toxicity of 3,3,5-TRIMETHYLCYCLOHEXYL ACRYLATE (CAS 86178-38-3) to Pseudokirchneriella subcapitata was conducted in accordance with the OECD 201 Test Guideline and GLP requirements.

The 48h-EC50 based on growth rate inhibition was not determined for mathematical reasons as the maximum observed effect was 29.8%. As a conservative approach, it is considered equal to the highest tested concentration (0.59 mg/L, geometric mean of the measured concentrations) for chemical safety assessment.

The 48h-EC10 based on growth rate inhibition was determined to be equal to 0.43 mg/L (geometric mean of the measured concentrations).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.59 mg/L

EC10 or NOEC for freshwater algae:

0.43 mg/L