Reaction mass of m,alpha-dimethylstyrene and p,alpha-dimethylstyrene

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Aug 2018 - 26 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006, Annex 5 corrected 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

m,α-dimethylstyrene

EC Number:

214-394-9

EC Name:

m,α-dimethylstyrene

Cas Number:

1124-20-5

Molecular formula:

C10H12

IUPAC Name:

1-isopropenyl-3-methylbenzene

Constituent 2

Reference substance name:

p,α-dimethylstyrene

EC Number:

214-795-9

EC Name:

p,α-dimethylstyrene

Cas Number:

1195-32-0

Molecular formula:

C10H12

IUPAC Name:

1-isopropenyl-4-methylbenzene

Test material form:

liquid

Details on test material:

Appearance: Clear colourless liquid
Storage conditions: At room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method: 2.0 mL from the approximate centre of the test vessels at t=0 h, t=24 h and t=72 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling. Instead, samples were taken from one replicate of each treatment.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel of the WAF prepared at a loading rate of 4.6 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The batch of the test item was a clear colourless liquid with a purity of 99% which was not completely soluble in test medium at the loading rates initially prepared. All glassware used during the preparation of test solutions was closed with minimal headspace in order to minimize vaporization of the test item.
- Preparation of test solutions started with loading rates individually prepared in the range of 0.46 to 100 mg/L. A two-day period of gentle magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Stock culture medium: M1, according to NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”), formulated using tap-water purified by reverse osmosis.
- Pre-culture: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium and test medium: Adjusted M2, according to OECD 201, formulated using tap-water purified by reverse osmosis.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

23 - 24°C

pH:

At t=0 h: 7.3 - 7.4
At t=72 h: 7.4 - 8.0

Nominal and measured concentrations:

Nominal: WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L
Measured: The measured concentrations at the start of the test were 0.27, 0.60, 1.2, 2.1, 5.6 and 13 mg/L at the WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L, respectively. At the end of the test, the concentrations were measured to be at 8.5-38% of initial. Therefore, Time Weighted Average concentrations were calculated to be 0.14, 0.29, 0.76, 1.2, 3.5 and 6.6 mg/L, respectively. See 'Any other details on materials and methods' for the TWA calculation and 'Any other details on results' for details on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 40 mL glass vials, airtight closed with no headspace to prevent any loss of the test item due to volatilization.
- Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 213 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, Adjusted M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted M2 medium
- Intervals of water quality measurement: pH: at the beginning and at the end of the test, for all concentrations and the control. Temperature of medium: continuously in a temperature control vessel.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 92 to 94 µE.m^-2.s^-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the WAF prepared at a loading rate of 2.2 mg/L to observe for any abnormal appearance of the algae compared to the control.

TEST CONCENTRATIONS
- Range finding study test loading rates: nominal 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, ErC50 is estimated to be between loading rates of 1.0 and 10 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed July 2018)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.82 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.72-0.92 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.99 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.87-1.1 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.76 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.14 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Based on statistical significance

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 0.76 mg/L when compared to the control.
- A concentration-related increase of growth rate inhibition was found at all test concentrations, up to ≥96% inhibition at the two highest concentrations tested. Statistically significant growth rate inhibition was found at all test concentrations. However, growth rate inhibition was below 10% at the three lowest test concentrations and therefore considered biologically not relevant. Thus, the NOEC was set at a TWA concentration of 0.76 mg/L based on biological relevance.
- The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae. Hence, it can be stated that the presence of the algae had no effect on the test item concentrations in test medium throughout the test.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the Final Test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50 for growth rate inhibition (72h-ErC50) was 1.05 mg/L with a 95% confidence interval ranging from 1.04 to 1.06 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Statistical significance: Step-down Jonckheere-Terpstra Test, α=0.05, one-sided, smaller.
Calculation of ECx-values for inhibition of growth rates was based on a 3-parameter normal cumulative distribution function (CDF) using non-linear regression analysis with the percentages of growth rate inhibition versus the logarithms of the corresponding TWA concentrations of the test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Any other information on results incl. tables

Table 1: Final Test: Test Samples

Time of sampling [hours]		Loading rate (a) [mg/L]		Concentration analyzed [mg/L]		Relative to initial [%]
0		0		n.d.
		0.46		0.272
		1.00		0.598
		2.2		1.21
		4.6		2.10
		4.6 (b)		2.86
		10		5.64
		22		12.8
24		0		n.d.		n.a.
		0.46		0.142		52
		1.00		0.382		64
		2.2		0.876		72
		4.6		2.42		115
		4.6 (b)		2.13		75
		10		4.16		74
		22		11.1		86
72		0		n.d.		n.a.
		0.46		0.0931		34
		1.00		0.102		17
		2.2		0.456		38
		4.6		0.18 (c)		8.5
		4.6 (b)		0.15 (c)		2.6
		10		2.02		16
		22		1.44		11

(a)  A water accommodated fraction (WAF) prepared at the loading rate.

(b) Without algae.

(c) Estimated value, calculated by extrapolation of the calibration curve.

n.d. Not detected.

n.a. Not applicable.

Table 2: Growth Rate and Percentage Inhibition for the Total Test Period

Test item TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.786	0.0181	6
0.14	1.743	0.0020	3	2.4^#
0.29	1.719	0.0133	3	3.7^#
0.76	1.688	0.0243	3	5.5^#
1.2	0.170	0.2951	3	90*
3.5	0.080	0.1392	3	96*
6.6	0.000	0.0000	3	100*

* effect was statistically significant,^# effect was statistically significant however biologically not relevant (<10%).

Table 3: Growth Rate and Percentage Inhibition at Different Time Intervals 

Test item TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.658		1.957		1.742
0.14	3	1.418	14	2.004	-2.4	1.809	-3.8
0.29	3	1.108	33	2.099	-7.2	1.951	-12
0.76	3	0.801	52	2.307	-18	1.956	-12
1.2	3	0.037	98	0.086	96	0.388	78
3.5	3	0.000	100	0.000	100	0.241	86
6.6	3	0.000	100	0.000	100	0.000	100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 72h-ErC10 and 72h-ErC50 for growth rate inhibition were 0.82 and 0.99 mg/L, based on TWA concentrations. The 72h-NOEC for growth rate inhibition was <0.14 mg/L based on statistical significance and 0.76 mg/L based on biological relevance.

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to individually prepared Water Accommodated Fractions of the test item prepared at nominal loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L (3 replicates per concentration) and an untreated control (6 replicates). Measured concentrations decreased to 8.5 -38% of initial at the end of the exposure period, therefore Time Weighted Average concentrations were calculated to be 0.14, 0.29, 0.76, 1.2, 3.5 and 6.6 mg/L in WAFs prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L, respectively. A concentration-related increase of growth rate inhibition was observed at all test concentrations. The EC50 for growth rate inhibition (72h-ErC50) and 72h-ErC10 were 0.99 and 0.82 mg/L, respectively. The 72h-NOErC was <0.14 mg/L based on statistical significance and 0.76 mg/L based on biological relevance. The study met all validity criteria, and is considered reliable without restriction.