Reaction Mass of 4-tert-butyl-2-hydroxycyclohexyl methacrylate and 5-tert-butyl-2-hydroxycyclohexyl methacrylate

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:

11 June 2015 - 04 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Test vessels were incubated in a Sanyo Gallenkamp orbital incubator set to maintain 21-24 °C for up to 72 h. The flasks were provided with continuous lightingin the spectral range of 400-700 nm at an intensity of 5360 lux and gently shaken at ca. 100 rpm.Aliquots of test solutions were removed from test vessels at 24, 48 and 72 h to allow algal cell concetrations to be determined. Aliquots of test solution were placed in an Improved Neabauer counting chamber and the number of alagal cells present visualised under a compound light microscope.The pH of test solutions was determined at test initiation and at 72 h.

Vehicle:

no

Details on test solutions:

Based on the results of a preliminary study, the definitive test was conducted with test solutions prepared as dilutions from a saturated solution of TBE, producing test concetrations of 1, 3.2, 10, 32 and 100 % saturation.The saturated solution was prepared by adding TBE (100.23 mg) to algal medium (1 L) and ultrasonicating for 60 mins. An aliquot (ca. 600 mL) of this saturated solution wassiphoned from the flask, taking care to exclide undissolved test item, and used directly as the 100 % test solution. The remaining stock was serially diluted (2:6.25 v/v) with algal medium to produce the required test concetrations. The control sampe contained algal medium only.Three replicate flasks at each test concentration (6 for control) contained 100 mL of test solution and were each inoculated with ca. 9800 cells/mL P. subcapitata.

Test organisms (species):

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

Details on test organisms:

Freshwater green alag Pseudokirchneriella subcapitata (CCAP 278/4) was initially obtained from the Culture Collection of Algae and Protozoa (CCAP), Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll, UK on 06 May 2015.The alga was cultured in-house.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5-23.2 °C

pH:

Freshly prepared solutions: 7.94-8.04Expired solutions at 72 h: 8.21-9.42 pH did not deviate by more than 1.5 units throughout the test period.

Nominal and measured concentrations:

Nominal concetrations: 100, 32, 10, 3.2 and 1 % saturationMean measured concentrations (0 and 72 h): 27.6, 6.6, 2.1, 1.2, 0.4 mg/L, respectively.More information available in Table 3.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

Analytical samples taken at 0 h and 72 h indicated that concetrations of TBE were maintained at 81-120 % of their initial value over the test period - with the exception of the 3.2 % saturated solution which increased by 100 % over the 72 h period. The overall mean measured concetration of TBE were 0.4, 1.2, 2.1, 6.6 and 27.6 mg/L for the 1, 3.2 10, 32 and 100 % saturated solutions, respectively. TBE was not detected in the control samples. See Table 3 for individual analyte results.See Tables 4 and 5 for growth and yield data summaries.

Reported statistics and error estimates:

See Table 6

Table 3       Mean measured concentrations of TBE during the definitive test

Nominal concentration of TBE (% saturation)	Time (h)	Measured concentration (mg/L)	Mean measured concentration (mg/L)	Deviation of 72 h measured concentration from 0 h (±%)
Control	0	nd	nd	0
	72	nd
1	0	0.42	0.4	-19
	72	0.34
	72*	0.43*	0.43*	n/a
3.2	0	0.78	1.2	+100
	72	1.56
10	0	2.04	2.1	+2
	72	2.09
32	0	6.87	6.6	-8
	72	6.35
100	0	25.0	27.6	+20
	72	30.1
	72*	27.5*	27.5*	n/a

nd: not detected

* flask not inoculated with algae

Table 4       Mean cell concentrations at 0, 24, 48 and 72 h during the definitive test

Mean measured concentration of TBE (mg/L)	Replicate	Mean measured concentration (x10^5cells/mL)
		0 h	24 h	48 h	72 h
Control	I	0.10	0.60	2.58	10.85
	II	0.10	0.58	2.43	10.95
	III	0.10	0.70	2.48	10.10
	IV	0.10	0.73	2.98	11.40
	V	0.10	0.60	2.65	12.10
	VI	0.10	0.60	2.43	10.20
	Mean	0.10	0.64	2.59	10.93
0.4	I	0.10	0.63	2.50	12.50
	II	0.10	0.68	2.60	10.23
	III	0.10	0.63	2.58	11.50
	Mean	0.10	0.65	2.56	11.41
1.2	I	0.10	0.55	2.13	9.18
	II	0.10	0.60	1.85	9.38
	III	0.10	0.58	1.93	7.83
	Mean	0.10	0.58	1.97	8.80
2.1	I	0.10	0.48	1.78	6.03
	II	0.10	0.50	1.83	5.63
	III	0.10	0.50	1.70	4.65
	Mean	0.10	0.49	1.77	5.44
6.6	I	0.10	0.50	0.95	2.05
	II	0.10	0.43	0.73	1.95
	III	0.10	0.48	0.78	1.85
	Mean	0.10	0.47	0.82	1.95
27.6	I	0.10	0.13	0.40	0.75
	II	0.10	0.18	0.40	0.70
	III	0.10	0.15	0.20	0.60
	Mean	0.10	0.15	0.33	0.68

Table 5       Cell growth group mean values determined from daily cell counts during the definitive test

Growth function	Time (h)	Mean measured concentration of TBE (mg/L)
		Control	0.4	1.2	2.1	6.6	27.6
Mean cell concentration (x 10^5cells/mL)	0-24	0.64	0.65	0.58	0.49	0.47	0.15
	0-48	2.59	2.56	1.97	1.77	0.82	0.33
	0.72	10.93	11.41	8.80	5.44	1.95	0.68
Average specific growth rate (µave/day)	0-24	1.86	1.89	1.77	1.62	1.57	0.44
	0-48	1.64	1.63	1.50	1.45	1.06	0.59
	0-72	1.57	1.58	1.50	1.34	1.00	0.65
Yield	0-24	1.34	1.37	1.20	0.99	0.93	0.14
	0-48	6.23	6.16	4.68	4.18	1.81	0.59
	0-72	27.09	28.28	21.75	13.35	4.63	1.46

Table 6       EC and NOEC values during the definitve test

Growth function	Time (h)	ErC10/EyC10 (mg/L)	ErC20/EyC20 (mg/L)	ErC50/EyC50 (mg/L)	NOEC (mg/L)
Average specific growth rate (µave/day)	0-24	5.6 (3.0, 8.2)	8.3 (5.3, 11.3)	16.2 (12.7, 19.7)	1.2
	0-48	0.9 (nr)	2.4 (0.3, 4.4)	12.2 (7.3, 17.2)	0.4
	0-72	0.4 (nr)	1.2 (0.3, 2.2)	10.5 (6.7, 14.2	0.4
Yield	0-24	2.9 (0.6, 5.1)	4.6 (1.9, 7.3)	10.3 (6.6, 14.1)	1.2
	0-48	0.4 (0.1, 0.6)	0.8 (0.3, 1.2)	2.8 (1.9, 3.7)	0.4
	0-72	0.4 (0.1, 0.7)	0.7 (0.3, 1.1)	1.9 (1.3, 2.4)	0.4

Values in paranthesis are confidence limits

nr: not reported as confidence limits contained 0

Validity criteria fulfilled:

yes

Conclusions:

The 72 h ErC10, ErC20 and ErC50 values for average specific growth rate were estimated to be 0.4, 1.2 and 10.5 mg/L, respectively, based on mean measured concetrations of TBE under the test conditions.The 72 h EyC10, EyC20 and EyC50 values for yield were estimated to be 0.4, 0.7 and 1.9 mg/L, respectively, based on mean measured concetrations of TBE under the test conditions.The 72 h NOEC for both growth rate and yield was estimated to be 0.4 mg/L based on mean measured concetrations of TBE.

Executive summary:

This study was designed to determine the effects of TBE on the growth of P. subcapitata, a unicellular freshwater green alga, over a 72 h period. The study was performed to meet the requirements of OECD Guideline 210 (2011).

Based on the results of a preliminary study, the definitive test was conducted with solutions prepared as dilutions of a saturated solution (nominally 100 mg/L). As the test item is only partially soluble, the method of preparation was selected to maximize the solubility of TBE under test conditions.

The exposure concentrations were nominally; 1, 3.2, 10, 32 and 100 % saturation, which corresponded to mean measured concentrations of 0.4, 1.2, 2.1, 6.6 and 27.6 mg/L, respectively. Analytical samples were taken at 0 and 72 h and indicated that concentrations of TBE were maintained at 81 -120 % of their initial value over the test period (with the exception of the discrepant 3.2 % saturated solution which increased by 100 % over the 72 h period.)

The 72 h ErC10, ErC20 and ErC50 values for average specific growth rate were estimated to be 0.4, 1.2 and 10.5 mg/L, respectively, based on mean measured concetrations of TBE under the test conditions.

The 72 h EyC10, EyC20 and EyC50 values for yield were estimated to be 0.4, 0.7 and 1.9 mg/L, respectively, based on mean measured concetrations of TBE under the test conditions.

The 72 h NOEC for both growth rate and yield was estimated to be 0.4 mg/L based on mean measured concetrations of TBE.

The test met OECD Guideline 201 validity criteria and was therfore considered to be acceptable.

Description of key information

72 h ErC50 = 10.5 mg/L, 72 h ErC10 = 0.4 mg/L; OECD 201, 2011 (GLP); Knight (2016)

Key value for chemical safety assessment

EC50 for freshwater algae:

10.5 mg/L

EC10 or NOEC for freshwater algae:

0.4 mg/L

Additional information

The 72 h ErC50 and ErC10 values were estimated as 10.5 and 0.4 mg/L, respectively, in a GLP study concucted in accordance with OECD Guideline 201. (Knight, 2016)