2,4,6-trimethylphenol

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Apr - 27 Apr 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted 02 Oct 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Rheinland-Pfalz, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Fresh and aged samples of control and all treatment groups
- Sampling method: Content of the test item was measured weekly in the freshly prepared and in the used solutions in extra replicates without algae and daphnia. Samples were taken from the same batch of the solutions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of a stock solution by weighing the test substance and sonication in a respective volume of test media until complete dissolution.
- Controls: Water
- Test concentration separation factor: 2.2
- Evidence of undissolved material: No

Test organisms (species):

Daphnia magna

Details on test organisms:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of a stock solution by weighing the test substance and sonication in a respective volume of test media until complete dissolution
- Controls: Water
- Test concentration separation factor: 2.2
- Evidence of undissolved material: No

TEST ORGANISM
- Common name: Water flea
- Age at study initiation (mean and range, SD): Between 0 and 24 hours
- Method of breeding: The holding is performed similar to the method described in the OECD guideline. They were cultured in groups of a maximum of 18 daphnia (in 2.5 L medium).
- Source: Umweltbundesamt (Berlin)
- Feeding during test: Yes
- Food type: Unicellular green alga Desmodesmus subspicatus
- Amount: 0.15 - 0.20 mg org. carbon/animal/day
- Frequency: On each working day. On Fridays, the double amount of alga suspension was added into the test vessels

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

267 mg CaCO3/L

Test temperature:

19.1 - 21.7 °C

pH:

7.6 - 8.5

Dissolved oxygen:

8.4 - 9.8 mg/L

Nominal and measured concentrations:

Nominal: Control, 0.12, 0.27, 0.56, 1.22, 2.68, 5.61 mg/L
Time-weighted mean measured: Control, 0.10, 0.21, 0.45, 1.22, 2.67, 5.55 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 glass beakers per treatment group
- Type (delete if not applicable): open, loosely covered with perforated transparent plastic wrap
- Material, size, fill volume: Glass, 100 mL, 80 ± 5 mL test solution
- Aeration: None
- Renewal rate of test solution: Three times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: M4 Medium

OTHER TEST CONDITIONS
- Photoperiod: 16 h daylight / 8 h darkness
- Light intensity: 1058 – 1111 lux

EFFECT PARAMETERS MEASURED:
- Reproduction (at each medium renewal)
- Immobility (at each medium renewal)
- Abnormalities as ephippia, presence of males, presence of aborted eggs and dead offspring (at each medium renewal)
- Length (at the end of the study)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: Yes
- Test concentrations: Control, 0.1, 1.0 and 5.0 mg/L
- Results used to determine the conditions for the definitive study: The chosen test design based on the result of a non GLP pre-test (duration 21 days) under semi-static conditions and medium renewal on Monday, Wednesday and Friday using freshly prepared test solutions. Based on the stability results, the chosen semi-static test design can be considered suitable for determining a mean exposure concentration in the range of the nominal concentration.

Reference substance (positive control):

yes

Remarks:

Acute toxicity studies were conducted twice a year wit the reference substance 3,5-Dichlorphenol

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: based on initially introduced parent animals

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.2 mg/L

95% CI:

>= 0.95 - <= 1.41

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: based on initially introduced parent animals

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

2.09 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

- Behavioural abnormalities: No
- Observations on body length and weight: No difference between control and the treatment groups
- Mortality of control: No
- Abnormal responses: No
- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The 48 h-EC50 against 3,5-Dichlorphenol was determined as 2.04 mg/L

Reported statistics and error estimates:

For each endpoint, the NOEC, LOEC, and, if possible, the EC50 and EC10 was determined. The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.3.0. In the highest tested nominal concentration (5.61 mg/L), all daphnia were found dead on day 2. In the second highest nominal concentration (2.68 mg/L) only 3 daphnia survived until the end of the test. In the other treatments no significant immobility was observed.
In the third highest tested concentration of 1.22 mg/L (nominal), a slight, statistically significant decrease in the reproduction of the daphnia was observed. The decrease in the reproduction of the daphnia in the lower concentrations was not statistically significant compared to the control.
The slight immobility at the nominal concentrations 0.27 mg/L and 0.56 mg/L was not caused by toxicity of the test item because in the next higher concentration 1.22 mg/L no immobility was observed. A regression analysis with all tested concentrations would result in an implausible, much too low value for the EC10 for the endpoint immobility. Therefore, for the endpoint immobility, an additional statistical evaluation was performed only with the 3 highest nominal concentrations.

Table 1: Calculated time weighted average (TWA) test concentrations for 2,4,6-trimethylphenol

Nominal concentration (mg/L)	Sampling days	Measured concentration (mg/L) Start (new media)	Measured concentration (mg/L) End (old media)	Time Weighted Average (mg/L)
0.12	0 and 2 (old)	0.1	0.09	0.10
	5 (new) and 7 (old)	0.11	0.09
	14 (new) and 16 (old)	0.1	0.09
	19 (new) and 21 (old)	0.11	0.09
0.27	0 and 2 (old)	0.22	0.2	0.21
	5 (new) and 7 (old)	0.25	0.22
	14 (new) and 16 (old)	0.22	0.17
	19 (new) and 21 (old)	0.24	0.2
0.56	0 and 2 (old)	0.45	0.41	0.45
	5 (new) and 7 (old)	0.5	0.45
	14 (new) and 16 (old)	0.47	0.39
	19 (new) and 21 (old)	0.5	0.44
1.22	0 and 2 (old)	1.24	1.13	1.22
	5 (new) and 7 (old)	1.38	1.18
	14 (new) and 16 (old)	1.28	1.08
	19 (new) and 21 (old)	1.34	1.17
2.68	0 and 2 (old)	2.80	2.45	2.67
	5 (new) and 7 (old)	2.81	2.28
	14 (new) and 16 (old)	2.89	2.47
	19 (new) and 21 (old)	3.10	2.60
5.61	0 and 2 (old)	5.83	5.28	5.55
	5 (new) and 7 (old)	NA	NA
	14 (new) and 16 (old)	NA	NA
	19 (new) and 21 (old)	NA	NA
NA: Not sampled since all testing organisms were immobile at that time

 

Table 2 : Nominal and percent of nominal concentration for 2,4,6-trimethylphenol during the 21 d reproduction study

Nominal Concentra tion	% of Nominal Concentration
(mg/L)	0 d (new)	2 d (old)	5 d (new)	7 d (old)	14 d (new)	16 d (old)	19 d (new)	21 d (old)
Blank control	--	--	--	--	--	--	--	--
0.12	86	79	93	78	87	76	93	79
0.27	82	75	91	80	82	63	89	73
0.56	80	73	90	81	84	70	88	79
1.22	102	93	113	97	105	88	110	96
2.68	104	91	105	85	108	92	116	97
5.61	104	94	--	--	--	--	--	--

 

 

Table 3: Test organism offspring after 21 d of exposure to 2,4,6-trimethylphenol

Nominal Concentra tion Test Item (mg/L)	Blank Control	0.12	0.27	0.56	1.22	2.67	5.61
Replicate
1	61	32	M	59	44	M	M
2	69	35	M	48	53	2	M
3	48	49	56	53	54	M	M
4	46	56	62	53	45	M	M
5	59	37	55	M	52	M	M
6	54	75	52	55	57	M	M
7	57	63	28	57	48	M	M
8	64	41	63	52	35	1	M
9	57	61	87	59	49	M	M
10	92	52	71	64	46	1	M
Mean	61	50	59	56	48*	1*	--
SD	13	14	17	5	6	1	--
RSD	21.35	27.87	28.44	8.54	13.02	43.3	--
SD = standard deviation;
RSD = relative standard deviation = coefficient of variation
M = mortality of parent animal
* statistically significant

 

Table 4: Test Organism fecundity based on the Surviving Parent Animals after 21d of exposure to 2,4,6-trimethylphenol

Nominal Con centration (mg/L)	Total Offspring	Surviving Pa rent Animals	Mean Offspring per Surviving Parent Animal	Inhibition of Reproduction (%)
Blank Control	607	10	61	0
0.12	501	10	50	17##
0.27	474	8	59	2
0.56	500	9	56	8
1.22	483	10	48	20*
2.67	4	3	1	98*
5.61	0	0	--	--
* statistically significant
## not statistically significant, an inhibition of 17 % is within a normal range for this biological system and is within the range of dispersion in the blank (blank mean = 61, blank
standard deviation 13, blank RSD = 21.35 %, see Table 3).

 

Table 5: Test organisms' body length after 21 d exposure to

2,4,6-trimethylphenol

Nominal conc.	Blank control	0.12 mg/L	0.27 mg/L	0.56 mg/L	1.22 mg/L	2.68 mg/L	5.61 mg/L
Replicate No.	Length in mm
1	6	4	--	6	5	--	--
2	6	4	--	6	6	5	--
3	6	6	6	6	5	--	--
4	6	6	6	5	6	--	--
5	6	6	5	--	6	--	--
6	6	6	5	6	6	--	--
7	6	6	5	5	5	--	--
8	6	6	6	5	6	5	--
9	5	6	6	6	6	--	--
10	5	6	6	5	6	5	--
mean	5.8	5.6	5.6	5.6	5.7	5.0
-- Dead adults

 

Table 6: Validity criteria for OECD 211

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (female Daphnia) does not exceed 20% at the end of the test.	0 %	Yes
The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60 in the control.	61	Yes

 

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The 21 d NOEC (reproduciton) for 2,4,6-trimethylphenol was 0.45 mg/L based on time weighted average cocentration and under semi-static conditions.

Description of key information

NOEC (21 d): 0.45 mg/L (time weighted average, reproduction, Daphnia magna, OECD 211)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.45 mg/L

Additional information

One GLP study on the long-term toxicity to aquatic invertebrates is available for 2,4,6-trimethylphenol. The study was conducted according to OECD guideline 211 investigating the chronic toxicity of the test item to Daphnia magna under semi-static conditions. Daphnia were exposed to the nominal concentrations of 0.12, 0.27, 0.56, 1.22, 2.68, 5.61 mg/L and an untreated control for 21 days. The test item was dissolved in test water. Corresponding concentrations of the new and aged test solutions for the chosen exposure intervals ranged between 73 % and 116 % of nominal.

Therefore, the results were based on time weighted average concentrations. No mortality or abnormal behavior was observed in the control. However, for the treatments mortality occurred for the nominal concentrations 0.27 mg/L with 20%, for 0.56 mg/L with 10%, for 2.67 mg/L with 70% and for 5.61 mg/L wit 100%. The mean living offspring number per surviving adult daphnid was 61 for the control and 50, 59, 56, 48 and 1 for the tested concentrations. The study is valid according to OECD guideline 211. The 21-day NOEC for reproduction and survival of Daphnia magna was 0.45 mg/L (time weighted average, based on initially introduced parent animals) and 1.22 mg/L (time weighted average).