2-(1,1-dimethylethyl)-6-[[3-(1,1-dimethylethyl)-2-hydroxy-5-methylphenyl]methyl]-4-methylphenyl 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:

14 Mar - 17 Mar 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

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment. Number 23. Guidance on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Organisation for Economic Co-operation and Development, Paris

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L WAF and the control
- Sampling method: Samples were taken at exposure initiation (0 hour), 24 hours, and exposure termination (72 hours). Samples analyzed on day 0 were removed from the test solutions in the volumetric flasks prior to filling the individual test flasks. Samples analyzed at 24 and 72 hours of exposure were removed from a composite sample of replicate vessels for the treatment and control. Each sample was collected from the approximate midpoint of the test vessel.
- Sample storage conditions before analysis: Archive samples were also collected from the exposure solutions at 0, 24, and 72 hours and stored frozen for possible future analysis and were discarded if not analyzed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A water-accommodated fraction (WAF) was prepared at exposure initiation from a 100 mg/L stock solution by adding 0.2031 g (0.2001 g as active ingredient) of the test item to 2 L of AAP medium. After addition, the solution was observed to contain a layer of visible undissolved test substance on the surface and throughout the water column. The solution was mixed overnight using a magnetic stir plate and Teflon®-covered stir bar. The temperature during overnight mixing ranged from 23 to 24 °C. Following overnight mixing, the solution was observed to be clear and colorless with a large amount of visible undissolved test substance on the surface and throughout the water column. The stock solution was filtered through a 0.45-μm Whatman membrane filter. Following filtration, the resulting solution was observed to be clear and colorless with no visible undissolved test substance. This 100% of a 100 mg/L WAF was used as the high treatment level solution for this exposure and was used to prepare the 10 and 1.0% of 100 mg/L WAF exposure solutions.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: 1648
- Source: UTEX The Culture Collection of Algae at the University of Texas, Austin, USA
- Method of cultivation: The stock cultures were maintained within the following conditions from at least the last transfer: a shaking rate of 100 ± 10 rpm, a temperature of 24 ± 1 °C and continuous illumination at the surface of the medium sufficient to provide photosynthetically-active radiation (PAR) of 60 to 120 μE/m2/S, maintained within ± 15%, i.e., 60 to 80 μE/m2/S. Lighting was supplied by fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using an environmental chamber. The inoculum used to initiate the toxicity test with was taken from a stock culture that had been transferred to fresh medium four days before testing.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22 - 23 °C

pH:

6.4 - 6.7 (control)
6.6 - 6.8 (100 mg/L)

Conductivity:

89 - 91 µS/cm

Nominal and measured concentrations:

100% of a water-accommodated fraction (WAF) prepared from a 100 mg/L stock solution, resulting in 0.00018 mg/L measured concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks (All flasks were conditioned prior to use by rinsing with the appropriate exposure solution).
- Type: All test vessels were fitted with stainless steel caps which permit gas exchange
- Material, size, headspace, fill volume: glass, size: 250 mL, fill volume: 100 mL, headspace: 150 mL
- Aeration: no, but constantly shaken
- Initial cells density: 1.0 x 10E+04 cells/mL
- Control end cells density: 52.63 E+04 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 80 μE/m²/S, approximately equivalent to 4500 to 6600 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At each subsequent 24-hour interval, cell counts were conducted using a hemocytometer and compound microscope for the control and 100% of 100 mg/L WAF treatment level. Observations of the health of the algal cells were also made and recorded at each observation interval.

TEST CONCENTRATIONS
- Range finding study: yes

Reference substance (positive control):

yes

Remarks:

zinc chloride

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: Stimulation of growth was recorded at 100 mg/L WAF.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, observed to be clear and colorless with no visible undissolved test substance.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 0.083 mg Zn/L, 95% confidence intervals of 0.077 to 0.092 mg Zn/L.
- Other: Previous reference testing determined a mean EC50 value of 0.090 mg Zn/L for P. subcapitata.

Reported statistics and error estimates:

In order to evaluate the treatment effects, an Equal Variance Two-Sample t-Test (p ≤ 0.05) was used to compare the results of the limit test concentration to the results of the control for all endpoints to detect statistically significant inhibition at the limit test concentration, if present. Additionally, the percent inhibition of each endpoint as compared to the control data was determined. CETISTM version 1.8 was used to perform the statistical comparisons.

Table 1: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	53 times	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	19 %	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	2.4 %	yes

Table 2: Concentrations measured in the exposure solutions during the 72-hour exposure of Pseudokirchneriella subcapitata.

Nominal concentration (% of 100 mg/L WAF)	Measured Concentration (μg/L)			Time-Weighted Average Concentration (μg/L)a
	0 hours	24 hours	72 hours
Control	< 0.050^b	< 0.050	< 0.050	NAc
100	0.23	0.36/0.066d	< 0.050f/< 0.050df	0.18
QCe#1	0.0876	0.0853	0.0959
0.100	(87.6)	(85.3)	(95.9)
QCe#2	0.882	0.948	0.923
1.00	(88.2)	(94.8)	(92.3)
QCe#3	18.5	18.3	19.1
20.0	(92.3)	(91.7)	(95.3)

a Time-weighted average concentrations were calculated using actual analytical data and not the rounded (2 significant figures) data presented in this table.

b Concentrations expressed as less than values were below the method detection limit (MDL). The MDL is dependent upon the lowest concentration calibration standard used and the dilution factor of the controls (i.e., 0.0500 μg/L x 1.00 = 0.050 μg/L).

c NA = Not applicable.

d Result of the additional sample without algae present to determine biological uptake/degradation

e QC = Quality Control sample. Percent recovery for each QC sample is presented in parentheses.

f Values equivalent to 50% of the MDL were used to calculate the time-weighted concentrations for instances in which samples resulted in measured values below the MDL.

Table 3: Cell density of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure.

Time-Weighted Average Concentration (μg/L)	Replicate	Cell Density (× 10E+04 cells/mL)			72 Hour Percent Inhibitiona (b)
		Observation Interval (Hours)
		24	48	72
Control	A	3.50	13.25	62.25
	B	3.50	16.75	52.50
	C	2.88	16.75	46.50
	D	3.88	16.50	51.00
	E	2.75	10.25	51.25
	F	2.88	15.25	52.25
	Mean (SD)	3.23 (0.46)	14.79 (2.60)	52.63 (5.19)	NAc
0.18	A	6.50	21.00	63.25
	B	3.25	19.50	66.50
	C	5.25	17.25	74.50
	D	4.00	16.75	87.00
	E	3.75	20.50	58.25
	F	7.75	16.50	64.50
	Mean (SD)	5.08 (1.76)	18.58 (1.99)	69.00 (10.29)	- 31

a Percent inhibition compared to the control.

b Mean, standard deviation (SD) and percent inhibition are calculated from original raw data, not from the rounded values presented in this table.

c NA = Not Applicable.

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

ErC10/50 (72 h) > 100 mg/L (nominal, Pseudokirchneriella subcapitata, OECD 201)

ErC10/50 (72 h) > 0.00018 mg/L (measured TWA, Pseudokirchneriella subcapitata, OECD 201) 

Additional information

One experimental study is available investigating the toxicity of 2-(1,1-dimethylethyl)-6-[[3-(1,1-dimethylethyl)-2-hydroxy-5-methylphenyl]methyl]-4-methylphenyl acrylate (CAS 61167-58-6) to the freshwater algae Pseudokirchneriella subcapitata. The study was performed according to OECD 201 (GLP) as a limit test with a filtered test solution of 100 mg/L. An appropriate amount of the test item was added to test medium followed by an overnight stirring period. Following mixing, the solution was observed to be clear and colorless with a large amount of visible undissolved test substance on the surface and throughout the water column. The stock solution was filtered through a 0.45 μm Whatman membrane filter. Following filtration, the resulting solution was observed to be clear and colorless with no visible undissolved test substance. The actual test concentration in the exposure vessels was determined by LC-MS analysis after 0, 24 and 72 h. The effect concentrations were based on the time-weighted-average (TWA) concentrations as well as the nominal concentrations. No inhibition of growth was recorded after 72 h and the ErC10/50 were reported as > 100 mg/L (nominal) and > 0.00018 mg/L (measured TWA).