2,4,6-trimethylphenol

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 May - 09 Jun 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

adopted 26 Jul 2013

Deviations:

yes

Remarks:

Temperature was not continuously measured in one test vessel, but in the storage tank. The exact count of the surviving fish was done at the end of the test. Due to the small size of the fish, an accurate count during the test was not possible.

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Rheinland-Pfalz, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.00 (control), 0.06, 0.12 mg/L
- Sampling method: Samples of each tested concentration including the control were taken on days 1, 5, 12, 20,27,34; during day 0 additional samples were taken directly from the aquarium and the inlet of the aquarium.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions with the concentration 122 mg/L test item were prepared in test medium. The test item was weighed in a glass vessel and transferred to a volumetric flask. Then the volumetric flask was filled with the test medium. The stock solution was sonicated with an ultrasonic bath until complete dissolution was reached. This stock solution was used for the preparation of the lower concentrated stock solutions at the concentrations of 1.22 mg/L and 0.61 mg/L test item including impurities. These stock solutions were mixed with test medium in the flow-through apparatus again. The mixture flew directly into the aquaria. Flow of stock solution and test medium were adjusted to reach the planned concentration of 0.12 mg/L and 0.06 mg/L test item in the treatments. At the beginning of the test, the test concentrations in the aquaria were prepared by the direct addition of a stock solution with the concentration 122 mg/L test item.
- Controls: Water
- Test concentration separation factor: 2 (extended limit test)
- Evidence of undissolved material: No

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Westaquarium
- Source: University Heidelberg (2019)
- Age at study initiation: Fertilized eggs (24 h). The age of the fish from which the eggs used in this study originated was 1 -2 years.
- Method of breeding: Fish were kept in a recirculation system using reconstituted freshwater. They were kept in groups of 6-8 fish per tank and separated into males and females.
- Feeding during test: The fish hatched after 4 days. Feeding was started 2 days after hatching on day 6. The amount of food depends on the age of the fish.
- Food type: From day 6 powdered flake food (GEMMA Micro 75); From day 20 until the end of the test Artemia nauplii
- Amount: ad libitum
- Frequency: powdered flake food 1-2 times per day; Artemia nauplii once a day, with the exception of day 23 - 26, 30 and day 31

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

35 d

Hardness:

53.4 mg CaCO3/L

Test temperature:

25.1 - 26.4 °C

pH:

7.2 - 7.6

Dissolved oxygen:

7.0 - 7.9 mg/L

Nominal and measured concentrations:

Nominal: 0.00, 0.07 and 0.12 mg test item/L
Measured :
- day 0 : - day 1 (aquaria): - day 5 (aquaria): - day 12 (aquaria): - day 20 (aquaria): - day 27 (aquaria): - day 34 (aquaria):

Details on test conditions:

TEST SYSTEM
- Embryo cups: spawn dishes
- Test vessel: aquaria
- Type: closed
- Material, size, headspace, fill volume: stainless steel, 46 x 15 x 23 cm, 12L
- Aeration: slightly aerated with aeration stones
- Type of flow-through: medium was added with flow meters. Stock solutions were added with tubing pumps
- Renewal rate of test solution: approx. two medium renewals per day
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Biomass loading rate: 12 - 34 mg/L (the loading rate was calculated by overall wet weight in each concentration at the end of the test divided by the volume of the test vessel of 12 L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted freshwater. Tap water was desalinated by reverse osmosis. The conductivity and pH value were then adjusted to a conductivity of 500 µS/cm and pH 7.5 by adding electrolyte solutions (sea salt and sodium bicarbonate).
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: 12h dark/12h light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Hatching, Survival, Appearance, Behaviour: once per working day
- Weight: at the end of the test, the surviving fish were weighed. Weighing was done in replicates because the single weights of fish are too low. Wet weight was determined.
- Length: at the end of the test.

RANGE-FINDING STUDY
- Test concentrations: Control, 0.1 and 1 mg/L test item
- Results used to determine the conditions for the definitive study: yes, at the concentration 1 mg/L the hatching rate and surviving rate was slightly lower than in the control. Whereas at the concentration 0.1 mg/L the hatchability and post-hatch survival rate were slightly higher than in the control.

POST-HATCH DETAILS
- Begin of post-hatch period: After 4 days
- No. of hatched eggs (alevins)/treatment released to the test chamber:
Control: Of 120 fish at the start 89 hatched
0.06 mg/L: Of 120 fish at the start 95 hatched
0.12 mg/L: Of 120 fish at the start 80 hatched

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.11 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Hatchability, Post hatch survival, wet weight, length

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages:
Surviving rate after 35 days:
Control: 94.4 %
0.06 mg/L: 93.7 %
0.12 mg/L: 87.5 %
- Days to hatch: starting at day 4
- Numbers hatched:
Control: Of 120 fish at the start 89 hatched
0.06 mg/L: Of 120 fish at the start 95 hatched
0.12 mg/L: Of 120 fish at the start 80 hatched
- Observations on body length and weight of young and/or exposed parents at one or more time periods:
Mean wet weight at the end of the test:
Control: 12.85 mg
0.06 mg/L: 11.12 mg
0.12 mg/L: 10.65 mg

Mean length at the end of the test:
Control: 11.62 mm
0.06 mg/L: 10.64 mm (statistically significant compared to the control but not biological relevant as the inhibition at the lowest concentration is based on a random variation and is not caused by toxicity of the test item. The inhibition in the higher concentration 0.12 mg/L of 5.8 % was identified as not statistically significant. 5.8% inhibition at the highest concentration is within the range of distribution in the blank control (mean 11.64, std. dev. 0.585, rel. std. dev. 5.0%).
0.12 mg/L: 10.95 mm
- Effect concentrations exceeding solubility of substance in test medium: No
- Incidents in the course of the test which might have influenced the results: No

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.0. In the statistical evaluation, a distinction must be made between quantal data (hatching rate and survival) and metric data (length and weight). For quantal data, a pre-test on the dose-response relationship was first performed to determine the NOEC. Based on the results of the pre-tests, a possible hypothesis test for the NOEC determination was selected by the software. With metric data, for determination of the NOEC, pre-tests for normal distribution, variance homogeneity and monotonic dose-response relationship were first performed. Based on the results of the pre-tests, a possible hypothesis test for the NOEC determination was selected by the software. For the endpoints hatching rate, survival rate and weight, the automatic mode of the program was successful and the NOEC determined by the automatically set test was plausible. With the present data for the length however, the automatic modus of the software failed and the NOEC must be determined by the user. For the length, the criteria of normal distribution and variance homogeneity were met, but the test for the dose-response relationship was not successful because in the higher concentration the fish were even slightly longer than in the lower concentration. Therefore, the test with the greatest statistical power, the Williams test, could not be applied for NOEC determination. The program therefore replaced the Williams test by the Dunnett test. However, the Dunnett test was also not applicable because the number of degrees of freedom was too low in this case. Therefore, no NOEC could be determined in automatic mode and a suitable test had to be selected by the user. For the Bonferroni-Holm-t test, all formal requirements were met.

Table 1: Nominal, measured and time weighted mean concentrations of 2,4,6,-trimethylphenol

Point in Time	Day 0*		Day 0**		Day 1		Day 5		Day 12		Day 20		Day 27		Day 34		Time weighted Mean concentration (mg/L)
Nominal Conc. mg/L	Measured Conc. mg/L	% of Nominal	Measured Conc. mg/L	% of Nominal	Measured Conc. mg/L	% of Nominal	Measured Conc. mg/L	% of Nominal	Measured Conc. mg/L	% of Nominal	Measured Conc. mg/L	% of Nominal	Measured Conc.	% of Nominal	Measured Conc.	% of Nominal
0.00	<LOD	NA	<LOD	NA	<LOD	NA	<LOD	NA	<LOD	NA	<LOD	NA	<LOD	NA	<LOD	NA
0.06	0.06	97%	0.09	155%	0.06	93%	0.06	101%	0.06	93%	0.08	126%	0.07	119%	0.07	115%	0.07
0.12	0.19	155%	0.12	102%	0.11	94%	0.11	94%	0.09	77%	0.11	93%	0.13	106%	0.12	97%	0.11

* sample from the aquarium

**sample from inlet to the aquarium

NA: not applicable

LOD: 0.02 mg/L

 

Table 2: Hatching Data after 35 days

Nominal Concentration in mg/L	Replicate	Number of Fish at the Start	Number of Fish hatched	Total Hatched	Hatching Rate
0	1	30	19	89	74.2 %
	2	30	17
	3	30	26
	4	30	27
0.06	1	30	25	95	79.2 %
	2	30	23
	3	30	20
	4	30	27
0.12	1	30	26	80	66.7 %
	2	30	19
	3	30	18
	4	30	17

Table 3: Mortality data after 35 days

Nominal Concentration in mg/L	Replicate	1)Number of Fish hatched	Dead Fish	Number of survived Fish at the End	Total Survived	Surviving Rate
0	1	19	3	16	84	94.4 %
	2	17	2	15
	3	26	0	26
	4	27	0	27
0.06	1	25	1	24	89	93.7 %
	2	23	3	20
	3	20	2	18
	4	27	0	27
0.12	1	26	0	26	70	87.5 %
	2	19	2	17
	3	18	4	14
	4	17	4	13

1) Number of survived fish at the end + Dead fish = Number of Fish hatched

Table 4: Weight of Surviving Fish after 35 days

Nominal Concentration in mg/L	Replicate	Mean Weight Fish per Replicate in mg	Total Mean
0	1	10.72	12.85
	2	15.24
	3	10.20
	4	15.23
0.06	1	13.48	11.12
	2	9.77
	3	10
	4	11.22
0.12	1	10.35	10.65
	2	9.88
	3	11.2
	4	11.15

Table 5: Length of Surviving Fish after 35 days

Nominal Concentration in mg/L	Replicate	Length per Fish in mm	Mean	Sd	CV
0	1	11.25	11.62	0.58	5 %
	2	12.13
	3	11.00
	4	12.11
0.06	1	10.58	10.64*	0.47	4.4%
	2	10.70
	3	11.22
	4	10.07
0.12	1	10.62	10.95	0.96	8.7%
	2	9.88
	3	11.14
	4	12.15

* statistically significant compared to the control but not biological relevant. For further information please see ‘Details on results'.

Table 6: Validity criteria for OECD 210

Criterion from the guideline	Outcome	Validity criterion fulfilled
The dissolved oxygen concentration should be >60% of the air saturation value throughout the test.	88.8 %	Yes
The water temperature should not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species (Annex 2).	0.06 mg/L: 93 % – 155 % of the nominal concentration 0.12 mg/L: 77 % - 155 % of the nominal concentration	Yes
The analytical measure of the test concentrations is compulsory.	Measurements accomplished	Yes
Overall survival of fertilised eggs and post-hatch success in the controls and, where relevant, in the solvent controls should be greater than or equal to the limits defined in Annex 2.	Hatching success in the Control: 74 %   Post-hatch success in the 94 %	Yes

Validity criteria fulfilled:

yes

Remarks:

For details please refer to field "any other information on results incl. tables"

Conclusions:

The 35 d NOEC (for all assessed endpoints) for 2,4,6-trimethylphenol was ≥0.11 mg/L based on time weighted mean cocentration and under flow-through conditions.

Description of key information

NOEC (35 d): ≥ 0.11 mg/L (time weighted mean measured, hatchability, post hatch survival, wet weight and length, Danio rerio, freshwater, OECD 210)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

>= 0.11 mg/L

Additional information

One study is available for 2,4,6-trimethylphenol investigating the long-term toxicity to fish according to OECD 210. The 35-day study was conducted under GLP with flow-through conditions and Danio rerio as test organism. The test organisms were exposed to nominal test concentrations of 0.00, 0.06, 0.12 mg/L. These test concentrations were analytical verified with GC/MS at days 0, 1, 5, 12, 20, 27 and 34 of the test. The measured concentrations lay between 77 % and 155 % of the nominal concentration. Therefore, the calculation/determination of the effect concentration was based on the time weighted mean of measured concentrations. No effects were observed during the test duration. The reported 35 d-NOEC was ≥ 0.11 mg/L for all investigated endpoints (hatchability, post hatch survival, wet weight and length) and was based on time weighted mean measured concentrations.