2,4,6-trimethylphenol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-trimethylphenol

EC Number:

208-419-2

EC Name:

2,4,6-trimethylphenol

Cas Number:

527-60-6

Molecular formula:

C9H12O

IUPAC Name:

2,4,6-trimethylphenol

Test material form:

solid: crystalline

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples (50 mL) for chemical analyses were collected from the control and all test concentrations used in the range finding test at the beginning and end of the 96-hours test period. However, based on the results of the toxicological tests, only the control and the concentrations bracketing the estimated EC50 were analyzed.
During the definitive test, 50-mL samples were collected from the control, lowest, two middle and highest test concentrations at 0 and 96 hours. At 96-hours only, the 50-mL sample was a subsample of pooled replicates. All samples were refrigerated and shipped on dry ice in sealed 50-mL polypropylene centrifuge tubes to Reimer Analytical & Associates (RAA) for analysis.

Test solutions

Vehicle:

no

Details on test solutions:

All stock solutions and exposure concentrations were prepared as product, which contains 87.72 % active ingredient. The definitive results are reported using the analytical results for the measured concentrations and interpolated values for the concentrations that were not measured. All values were then time-weighted from 0 to 96 hours.

For the range-finding test, a stock solution of approximately 800 mg product/L was prepared by mixing 401.4 mg of the test product with 500 mL of nutrient solution. The solution was then placed on a magnetic stir plate and agitated for 20 hours and 45 minutes. For the definitive test, a stock solution of approximately 80 mg product/L was prepared by mixing 40.7 mg of test product with 500 mL of nutrient solution. The solution was then placed on a magnetic stir plate and agitated for 22.5 hours.

All stock solutions were prepared approximately 24 hours in advance of the test initiation to allow the solution to reach equilibrium. The majority of the test substance appeared to be readily soluble in water. However, fine particulates were observed in the stock solutions during both the range-finding and definitive tests.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Source: Uni-algal cultures of Selenastrum capricornutum Printz 1913 (original source UTCC #37)
- Age of inoculum (at test initiation): 3-7 days

ACCLIMATION
- Culturing media and conditions: Algal growth medium (liquid growth medium recommended by Environment Canada 1992); axenically subcultred into fresh medium twice a week.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Test temperature:

24 °C

pH:

7.24 - 7.61

Nominal and measured concentrations:

Nnominal concentrations: 0, 0.82, 2.05, 5.12, 12.8, 32.0, and 80.0 mg/L
Measured interpolated concentrations: 0, 0.55, 1.61, 3.43, 9.21, 23.46, and 58.19 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Clear glass 250 mL Erlenmeyer flasks covered with Jaece non-toxic foam plugs
- Material, size, headspace, fill volume: 50 mL test solution
- Initial cells density: 1E4 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes, algal growth medium (liquid growth medium recommended by Environment Canada 1992)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Algal growth medium (liquid growth medium recommended by Environment Canada 1992); filter sterilzed, but otherwise treated in a non-axenic manner.
- Intervals of water quality measurement: pH as measured at the start and end of the test in all concentrations.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continous
- Light quality: Cool-white fluorescent
- Light intensity: Measured at the surface of the liquid in the flasks. 4 ± 10 % kLux for culturing; 8 ± 20 % KLux for testing.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell number measured daily using a haemocytometer

TEST CONCENTRATIONS
- Range finding study: Yes
- Range finding test nominal concentrations: 0, 0.08, 0.8, 8, 80, and 800 mg/L

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

6.03 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 5.38-6.58

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.59 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 5.15-6.07

Effect concentrations 3

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

8.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 7.76-9.37

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 7.44-8.73

Effect concentrations 5

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.61 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.61 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

CHEMICAL CONFIRMATION
Samples of the control, low, two middle and high exposure concentrations of the test substance were analyzed for the active ingredient, to confirm the nominal test concentrations. The average recovery of the observed active ingredient concentrations in spiked aqueous solutions was 96 ± 2 %, demonstrating acceptable method accuracy. The prevent recovery (measured/nominal) was greater than 80%. the difference between 0- and 96-hour test solutions was greater than 20%.

RANGE-FINDING TEST
Results from the range finding test indicated that the 50% inhibitory effect level was between 0.7 to 7.0 mg/L (nominal, active ingredient, is equivalent to 0.8 and 80 mg product/L) for 72- and 96-hour cell number and area under the growth curve. For 72-hour growth rate, the 7.0 mg active ingredient/L (8.0 mg product/L) concentration elicited a 58.87% effect so the expected 50% inhibitory effect level was between 0.7 and 70.2 mg/L (0.8 and 80 mg product/L). The estimated 50% inhibitory effect level for 96-hour growth rate was between 7.0 and 70.2 mg/L (nominal, active ingredient, is equivalent to 8.0 and 80 mg product/L)

DEFINITIVE TEST
See any other information on results incl. tables.

Results with reference substance (positive control):

The EC25 was determined to be Based on routinely checks: The coefficient of variation (CV) for EC25s, based on cell number, for the last eight reference test based on S. capricornutum 96-hour cell number was 54.3%

Any other information on results incl. tables

Growth rate 0 - 96hours (x10,000)

Nominal conc. (mg/L)	Measured Interpolated Conc. (mg/L)	Mean	Stdev	% Inhibition
Control	0.00	0.05998	0.00089
0.82	0.55	0.05979	0.00089	0.32
2.05	1.61	0.05900	0.00065	1.63
5.12	3.43	0.05123	0.00040	14.59
12.8	9.21	0.01403	0.00514	76.62
32	23.46	0.00610	0.00557	89.83
80	58.19	0.00595	0.01047	90.07

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 96-hour measured (TWA) EC50 and NOEC for growth rate were 6.03 and 1.61 mg/L, respectively.

Executive summary:

According to OECD 201 and in compliance with GLP, a growth inhibition test was performed on fresh water green alga (selenastrum capricornutum). A range-finding test was conducted (0, 0.08, 0.8, 8, 80, and 800 mg/L; as test product) to determine the test concentrations to be used for the definitive test. For the definitive test nominal concentrations were 0, 0.82, 2.05, 5.12, 12.8, 32.0, and 80.0 mg/L (4 replicates per concentration or control). In the definitive test, the test concentrations that were not analytically verified were interpolated from the measured test concentrations. All concentrations were then time weighted (OECD, 1998b). Based on the time-weighted concentrations, the 96-hour ECr50 is 6.03 mg/L and the 96-hour NOECr is 1.61 mg/L. Based on the nominal concentrations, the 96-hour ECr50 is 8.64 mg/L and the NOECr is 2.05 mg/L.