2,4,6-trimethylphenol

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

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-hour test period. However, based on the results of the toxicological tests, only the control and the concentrations immediately bracketing the estimated LC50 were sent for chemical verification.
At the start and end of the definitive test, samples from each replicate in the control, lowest, middle and highest test solutions were pooled and a 50-mL sub-sample saved for analyses. All samples were refrigerated prior to shipping 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 dosed as product. For the range finding test, an 800 mg/L stock solution was prepared by mixing 14.4017 g of product into 18 L of groundwater. For the definitive test, 4 L of an 800 mg/L stock solution was prepared in two 2-L volumetric flasks by mixing 1.6008 g and 1.5999 g into each flask with groundwater. The two flasks containing the 800 mg/L stock solution were thoroughly homogenized prior to preparation of each test solution. All stock solutions were prepared approximately 24 hours in advance of 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):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Method of breeding and source: Rainbow trout used for testing were obtained as eyed eggs from a certified disease-free hatchery (Rainbow Springs Hatchery, Thamesford, Ontario). Eyed eggs were initially held in Heath Incubators. They were then transferred to fiberglass holding tanks of approximately 260 L capacity. Light was supplied by cool white fluorescent tubes on automatic timers which provide a 16-hour light / 8-hour dark photoperiod. Water temperature is maintained at a constant temperature of 15 ± 2 °C. Rainbow trout were fed commercial No. 1 pellet size trout food at a daily ration approximating 1-4 % wet body weight. Fish were weighed on a weekly basis to determine ration.
- Length at study initiation (fork length in mm) (mean ± 2 SD): 38.9 ± 5.9
- Weight at study initiation (g) (mean ± 2 SD): 0.5 ± 0.24
- Feeding during test: No

ACCLIMATION
- Acclimation period: 14-days
- Health during acclimation: Previous 7d stock tank mortality 0.96%

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

308 mg/L as CaCO3

Test temperature:

14 - 15 °C

pH:

6.8 - 7.1

Dissolved oxygen:

8.8 - 10.3 mg/L

Nominal and measured concentrations:

Nominal product concentrations: 0, 0.8, 1.7, 3.8, 8.3, 18.2, and 40 mg/L
Measured test concentrations active ingredient: 0, 0.7, 1.7, 3.7, 8.2, 17.2, and 37.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Material, size, headspace, fill volume: 15L
- Aeration: 6.5 ± 1 mL/L/min. dissolved oxygen> 60 % of saturation in control.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 0.33 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater (initial hardness approximately 308 mg/L as CaCO3; initial pH approximately 8.3) adjusted to approximately pH 7 prior to testing.
- Alkalinity: 5 mg/L as CaCO3
- Ca/mg ratio: 2.07
- Na/K ratio: 4.70
- Intervals of water quality measurement: pH, dissolved oxygen, conductivity, temperature at 0, 3, 24, 48, 72, and 96 hours

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16-hour light, 8-hour dark (with 30-minute transition period)
- Light intensity and quality: Ambient laboratory illumination, 100 to 500 lux

EFFECT PARAMETERS MEASURED: Mortality, stressed behaviour visual observations at 0, 3, 24, 48, 72, and 96 hours.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations:
Nominal product concentrations: 0, 0.08, 0.8, 8, 80, and 800 mg/L
Nominal active ingredient concentrations: 0, 0.07, 0.7, 7, 70, and 702 mg/L

Reference substance (positive control):

yes

Remarks:

Phenol

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

9.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 3.7 - 17.2

Details on results:

CHEMICAL CONFIRMATION
Samples of the control, low, medium and high exposure concentrations of the test substance were analyzed for the active ingredient, to confirm the nominal definitive test concentrations. The average recovery of the active ingredient observed concentrations in spiked aqueous solutions was 96 %, demonstrating acceptable method accuracy.
Measured active ingredient concentrations were within 20 % of nominal concentrations. However, in two cases, the difference between nominal and measured concentrations was 19 %. Furthermore, percent differences between old and new test solutions were also greater than 20 %. Based on these results, measured concentrations were used in endpoint calculations. Exposure concentrations that were not analytically verified were interpolated from the measured concentrations. All concentrations were then time weighted (OECD, 1998b).

RANGE-FINDING TEST
The test met all validity criteria. Results from the range-finding test indicated that the 96-hour LC50 was in the range of 7 to 70 mg/L (nominal, as active ingredient) for rainbow trout.

DEFINITIVE TEST
The test met all validity criteria. The 96-hour LC50 was determined to be 9.7 mg/L (95 % confidence limits of 3.7 to 17.2 mg/L) based on time-weighted, measured and interpolated concentrations of the active ingredient. See any other information on results incl. tables

Results with reference substance (positive control):

Phenol was the reference toxicant used in this study. The LC50 was determined to be 8.4 mg/L. The coefficient of variation (CV) for LC50 values for rainbow trout was 11.4 %.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Test conc. Nominal product	Test conc. Measured active ingredient**	Mortality
		A	B	A	B	A	B	A	B	A	B	A	B
		0 hours*		3 hours		24 hours		48 hours		72 hours		96 hours
40	37.5	0	0	10	10	10	10	10	10	10	10	10	10
18.2	17.2	0	0	10	10	10	10	10	10	10	10	10	10
8.3	8.2	0	0	0	0	2	3	3	3	3	3	3	3
3.8	3.7	0	0	0	0	0	0	0	0	0	0	0	0
1.7	1.7	0	0	0	0	0	0	0	0	0	0	0	0
0.8	0.7	0	0	0	0	0	0	0	0	0	0	0	0
control	control	0	0	0	0	0	0	0	0	0	0	0	0

*All fish in 8.3, 18.2 and 40 mg/L concentration were immobile within 5 minutes of exposure

** Based on time-weighted measured values (and interpolated values for concentrations not measured)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions the 96-hour, measured (TWA) LC50 for the active ingredient is 9.7 mg/L.

Executive summary:

According to OECD 203 and in compliance with GLP, a fish acute toxicity test was conducted using rainbow trout (Oncorhynchus mykiss). A range-finding test was conducted to determine the test concentrations to be used for the definitive test. Exposure concentrations, one order of magnitude apart (800, 80, 8, 0.8 and 0.08 mg/L; as test product), and a control were used. The preliminary estimates of the LC50 for 96-hour rainbow trout mortality was between 8 and 80 mg/L, as product (or 7 to 70 mg/Lnominal 2,4,6— trimethylphenol). From these results, the following nominal concentrations were chosen for the definitive test: 40.0, 18.2, 8.3, 3.8, 1.7 and 0.8 mg/L, plus controls. In the definitive rainbow trout study, 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 LC50 for rainbow trout mortality was 9.7 mg/L (with 95 % confidence limits of 3.7 to 17.2 mg/L).