Reaction mass of 4-(2-methylbutan-2-yl)phenol and 2,4-bis(2-methylbutan-2-yl)phenol

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 December 2015 to 25 January 2016

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:

yes

Remarks:

Cell densities were measured at a wavelength of 680 nm instead of 720 nm. Evaluation: The chosen wavelength conforms to the wavelength range recommended in OECD guideline 201.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch OP: C605E003.1
Study specific test item information
Purity/composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum-foil. To liquefied and homogenize: heat the test item until maximum 130°C under nitrogen.Stability at higher temperatures: Yes, maximum temperature: 130°C maximum duration: multiple hours if stored under nitrogen. If heated only minutes
Chemical name (IUPAC), synonym or trade name: Phenol, 1,1-dimethylpropyl derivs.CAS Number: 1639131-79-5
Molecular structure: Reaction mass so not available
Molecular formula: Reaction mass so not available
Molecular weight: Reaction mass so not available
Highly reactive to water: Not indicated by sponsor
Volatile: 20°C ≤ 1.9 Pa = ≤ 1.4 x 10-2 mm Hg; 25°C ≤ 3.1 Pa = ≤ 2.3 x 10-2 mm Hg (determined at WIL Research Europe, project 509995)

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all WAFs and the control according to the schedule below.Frequency: at t=0 h, t=24 h and t=72 hVolume: 2.0 mL
Storage: Samples were stored in a freezer until analysis.At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration 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.Additionally, reserve samples of 2.0 mL were taken from all WAFs and the control for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

Details on test solutions:

The batch of Phenol, 1,1-dimethylpropyl derivs. tested was a colourless to pale yellow solid of unknown or variable composition. No correction was made for the composition of the test item.Handling of test item and preparation of test solutions was performed under dimmed light.The test item was heated in a water bath (90°C) to liquefy and homogenize. Sub stocks were weighed for several projects. These sub stocks were stored under nitrogen in the dark and re-liquefied (100°C) and homogenized before use.All test solutions were prepared separately at different loading rates applying three days of magnetic stirring to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for 1-1.5 hours where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used for testing.After preparation, volumes of 50 mL were added to each replicate of the respective test concentration.Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.Fresh water algae culture
Stock culture: 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.
Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis.
Pre-culture: 3 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 104 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: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C.

pH:

7.9 - 8.1

Dissolved oxygen:

Not specified in the study report.

Salinity:

Not applicable - Freshwater study

Conductivity:

Not specified in the study report.

Nominal and measured concentrations:

The average exposure concentrations were calculated being the Time Weighted Average (TWA) of the concentrations of Phenol, 1,1-dimethylpropyl derivs. (major component) measured in the samples taken at the start (Ct=0), after 24 hours (Ct=24) and the end of the test (Ct=72).

Details on test conditions:

Combined limit/range-finding test
The study started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a WAF prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions
:-Three replicates per concentration were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L
;-Cell density was determined on 24-hour intervals in the control and the 100 mg/L WAF. In the WAFs prepared lower loading rates cell density was determined only at the end of the test
:-pH was only measured in the control and the 100 mg/L WAF
;-At the end of the test algae were not observed to verify a normal and healthy appearance
;-In addition to the samples taken from the test medium, the residue left after siphoning of the 100 mg/L WAF was kept for possible analysis.

Final test
Test concentrations
Phenol, 1,1-dimethylpropyl derivs.: WAFs prepared at loading rates of 0.2, 0.6, 2.0, 6.0 and 20 mg/L
Control: Test medium without test item or other additives
Replicates: 3 replicates of each WAF; 6 replicates of the control; 1 extra replicate of the control and each WAF for sampling purposes; 1 or 2 replicates of each WAF without algae.
Test procedures and conditions
Test duration: 72 hoursTest type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 86 to 92 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
Measurements
pH: At the beginning and at the end of the test.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the final test microscopic observations were performed on the 2.0 mg/L WAF to observe for any abnormal appearance of the algae.
Recording of cell densities
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 immersion probe (pathlength =20 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.56 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Details on results:

Combined limit/range-finding test
The expected EC50 for inhibition of both growth rate and yield was between concentrations obtained in WAFs prepared at loading rates of 1.0 and 10 mg/L.All three WAFs were analysed. Quantification was based on the peak area of the major component, as described in WIL Research Project 509995. The initial concentration in the WAFs prepared at 1.0, 10 and 100 mg/L was 0.86, 10 and 97 mg/L, respectively. These concentrations remained stable during the first 24 hours of exposure (92-97% of initial). At the end of the 72-hour test period the lowest concentration had decreased to 78% of initial, while the two higher concentrations were still in agreement with initial (81-84%).

All test conditions were maintained within the limits prescribed by the study plan.Final testAll WAFs were analysed. Quantification was based on the peak area of the major component, as described in WIL Research Project 509995. The initial concentration in the WAFs prepared at 0.2, 0.6, 2.0, 6.0 and 20 mg/L was 0.22, 0.59, 2.2, 6.1 and 20 mg/L, respectively. These concentrations remained stable during the first 24 hours of exposure (90-101% of initial). At the end of the 72-hour test period the lowest concentration had decreased to 78% of initial, while all higher concentrations were still in agreement with initial (83-91%). Samples taken from the 2.0 mg/L WAF with and without algae showed similar concentrations during the test period. Based on these results, the average exposure concentrations were calculated.Inhibition of growth rate and inhibition of yieldInhibition of growth rate increased with increasing concentration of Phenol, 1,1-dimethylpropyl derivs. from 0.20 mg/L upwards resulting in 97% inhibition at a TWA concentration of 18 mg/L. Statistically significant inhibition of growth rate was found at test concentrations of 0.20 mg/L and higher. However, biologically significant inhibition (≥10% inhibition of growth rate) was observed only at test concentrations of 2.1 mg/L and higher.Inhibition of yield increased with increasing concentration of Phenol, 1,1-dimethylpropyl derivs. From 0.20 mg/L upwards resulting in >95% inhibition at and above a TWA concentration of 5.8 mg/L. Statistically significant inhibition of yield was found at test concentrations of 0.20 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.Experimental conditionsThe pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

Results with reference substance (positive control):

Potassium dichromate inhibited the growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher.The EC50 for growth rate inhibition (72h-ERC50) was 1.5 mg/l with a 95% confidence interval ranging from 1.4 to 1.6 mg/l. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/l. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.The EC50 for yield decrease (72h-EYC50) was 0.51 mg/l with a 95% confidence interval ranging from 0.49 to 0.53 mg/l. The historical ranges for yield decrease lie between 0.43 and 1.1 mg/l. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Detailed in table form - see Overall remarks, attachments for information.

Mean cell densities (x10⁴cells/mL) during the combined limit/range-finding test

Time (h)	Phenol, 1,1-dimethypropyl dervis. WAF prepared at the given loading rate (mg/L)
	Control	1.0	10	100
0 24 48 72	1.0 8.2 50.8 253.9	1.0 - - 205.6	1.0 - - 2.0	1.0 1.6 1.7 1.1

- Not measured

 

Percentage inhibition of growth rate during the combined limit/range-finding test

Phenol, 1,1-dimethylpropyl dervis. WAF prepared at the given loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control 1.0 10 100	1.845 1.775 0.223 0.030	0.0176 0.0239 0.0589 0.0389	6 3 3 6	3.8 87.9 98.4

 

Percentage inhibition of yield during the combined limit/range-finding test

Phenol, 1,1-dimethylpropyl dervis. WAF prepared at the given loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control 1.0 10 100	252.9 204.6 1.0 0.1	13.62 14.83 0.33 0.13	6 3 3 6	19.1 99.6 100.0

 

Measured concentrations versus loading rates

Phenol, 1,1-dimethylpropyl dervis. WAF prepared at the given loading rate (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72h
0.2 0.6 2.0 2.0^1 6.0 20	0.219 0.590 2.15 2.19 6.12 20.2	0.215 0.595 2.14 2.20 5.92 18.2	0.170 0.488 1.93 1.91 5.57 17.8	0.20 0.56 2.1 2.1 5.8 18

¹without algae

 

Percentage inhibition of growth rate (total test period) during the final test

TWA conc. Phenol, 1,1-dimethylpropyl dervis. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control 0.20 0.56 2.1 5.8 18	1.880 1.816 1.810 1.475 0.831 0.063	0.0383 0.0352 0.0545 0.0268 0.2383 0.0613	6 3 3 3 3 3	3.4^# 3.7^# 21.5^* 55.8^* 96.6^*

^*- effect was statistically significant

^#- effect was statistically significant but biologically insignificant (<10%)

 

Percentage inhibition of growth rate at different time intervals during the final test

TWA conc. Phenol, 1,1-dimethylpropyl dervis. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72 h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control 0.20 0.56 2.1 5.8 18	6 3 3 3 3 3	2.364 2.282 2.251 2.075 1.769 0.860	0.0 3.5 4.8 12.2 25.2 63.6	1.671 1.488 1.599 1.047 0.158 -0.295	0.0 10.9 4.3 37.3 90.6 117.7	1.604 1.678 1.579 1.305 0.565 -0.375	0.0 -4.6 1.6 18.7 64.8 123.4

 

Percentage inhibition of yield during the final test

TWA conc. Phenol, 1,1-dimethylpropyl dervis. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control 0.20 0.56 2.1 5.8 18	281.7 232.4 229.0 82.8 13.4 0.2	33.77 24.30 39.19 6.89 10.51 0.23	6 3 3 3 3 3	17.5^* 18.7^* 70.6^* 95.3^* 99.9^*

^*- effect was statistically significant

 

pH levels recorded during the final test

TWA conc. Phenol, 1,1-dimethylpropyl dervis. (mg/L)	pH
	t=0h	t=72h
Control 0.20 0.56 2.1 5.8 18	8.1 8.1 8.1 8.1 8.1 8.1	7.9 7.9 7.9 7.9 7.9 7.9

 

Overview of % inhibition of growth rate in the reference test

Nominal conc. K2Cr2O7(mg/L)	Mean	Std. Dev.	n	%Inhibition
Control 0.18 0.32 0.56 1.0 1.8 3.2	1.661 1.649 1.594 1.383 1.027 0.731 0.392	0.0979 0.0314 0.0414 0.0139 0.0765 0.1871 0.0076	3 3 3 3 3 3 3	0.7 4.0 16.8 38.2 56.0 76.4

 

Overview of % decrease of yield in the reference test

Nominal conc. K2Cr2O7(mg/L)	Mean	Std. Dev.	n	%Decrease
Control 0.18 0.32 0.56 1.0 1.8 3.2	149.0 140.0 118.8 62.3 21.2 9.0 2.3	43.35 13.38 15.08 2.67 5.36 6.15 0.07	3 3 3 3 3 3 3	6.0 20.3 58.2 85.8 93.9 98.5

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for growth rate inhibition (72h-ERC50) was 4.7 mg/L with a 95% confidence interval ranging from 4.2 to 5.2 mg/L.The 72h-NOEC for growth rate inhibition was 0.56 mg/L.The EC50 for yield inhibition (72h-EYC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.0 to 1.3 mg/L.The 72h-NOEC for yield inhibition was <0.20 mg/L.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Phenol, 1,1-dimethylpropyl derivs..

 

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 and the OECD series on testing and assessment number 23, 2000.

 

The batch of Phenol, 1,1-dimethylpropyl derivs. tested was a colourless to pale yellow solid of unknown or variable composition. No correction was made for the composition of the test item.

Handling of test item and preparation of test solutions was performed under dimmed light.

The test item was heated in a water bath (90°C) to liquefy and homogenize. Sub stocks were weighed for several projects. These sub stocks were stored under nitrogen in the dark and re-liquefied (100°C) and homogenized before use.

 

All test solutions were prepared separately at different loading rates applying three days of magnetic stirring to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixtures were left to stabilize for 1-1.5 hours where after the clear and colourless Water Accommodated Fractions (WAFs) were siphoned out and used for testing.

 

Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 0.2, 0.6, 2.0, 6.0 and 20 mg Phenol, 1,1-dimethylpropyl derivs. per litre. The initial cell density was 104 cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

 

All WAFs were analysed. Quantification was based on the peak area of the major component, as described in WIL Research Project 509995. The initial concentration in the WAFs prepared at 0.2, 0.6, 2.0, 6.0 and 20 mg/L was 0.22, 0.59, 2.2, 6.1 and 20 mg/L, respectively. These concentrations remained stable during the first 24 hours of exposure (90-101% of initial). At the end of the 72-hour test period the lowest concentration had decreased to 78% of initial, while all higher concentrations were still in agreement with initial (83-91%). Samples taken from the 2.0 mg/L WAF with and without algae showed similar concentrations during the test period. Based on these results, the average exposure concentrations were calculated to be 0.20, 0.56, 2.1, 5.8 and 18 mg/L.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The effect parameters obtained in this study are summarized in the table below. The concentrations were related to the major component of Phenol, 1,1-dimethylpropyl derivs.

	Parameter (mg/L)	NOEC	EC10	EC20	EC50
Growth rate	Value Lower 95%-cl Upper 95%-cl	0.56	1.4 1.1 1.7	2.1 1.7 2.5	4.7 4.2 5.2
Yield	Value Lower 95%-cl Upper 95%-cl	<0.20	<0.20	0.46 0.38 0.54	1.2 1.0 1.3

 

In conclusion:

Phenol, 1,1-dimethylpropyl derivs. inhibited growth rate of this fresh water algae species significantly at a TWA concentration of 2.1 mg/L and higher. The EC50 for growth rate inhibition (72h-ERC50) was 4.7 mg/L with a 95% confidence interval ranging from 4.2 to 5.2 mg/L. The 72h-NOEC for growth rate inhibition was 0.56 mg/L.

Phenol, 1,1-dimethylpropyl derivs. inhibited the yield of this fresh water algae species significantly at all TWA concentrations tested. The EC50 for yield inhibition (72h-EYC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.0 to 1.3 mg/L. The 72h-NOEC for yield inhibition was <0.20 mg/L.

All concentrations were related to the major component of Phenol, 1,1-dimethylpropyl derivs.

Description of key information

Key values determined in a GLP accredited laboratory study performed in accordance with OECD Guideline 201 and EU Method C.3.

Phenol, 1,1-dimethylpropyl derivs. inhibited growth rate of this fresh water algae species significantly at a TWA concentration of 2.1 mg/L and higher. The EC50 for growth rate inhibition (72h-ERC50) was 4.7 mg/L with a 95% confidence interval ranging from 4.2 to 5.2 mg/L. The 72h-NOEC for growth rate inhibition was 0.56 mg/L.

Phenol, 1,1-dimethylpropyl derivs. inhibited the yield of this fresh water algae species significantly at all TWA concentrations tested. The EC50 for yield inhibition (72h-EYC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.0 to 1.3 mg/L. The 72h-NOEC for yield inhibition was <0.20 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.7 mg/L

EC10 or NOEC for freshwater algae:

0.56 mg/L