6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol

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:

01 Feb 2018. Day 28 post-hatch (test end) was 05 Mar 2018. Total test duration was 32 days.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

OECD Guideline study conducted to GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples (25 mL) from the control, solvent control and each test concentration were taken for analysis prior to egg addition and on Day 0 (pre-hatch) and Days 0, x, x and x (post-hatch). A single replicate from the controls and each test concentration were sampled on each occasion with replicates being sampled alternately

Vehicle:

yes

Details on test solutions:

Information provided by the Sponsor indicated that the water solubility of the test substance was <4 µg/L. Preliminary solubility work indicated that a colourless solution could be achieved at a concentration of 80 µg/L in treated mains water when spiked via an auxiliary solvent (dimethylformamide) at a solvent rate of 50 µL/L.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The Pimephales promelas eggs used in the early life stage test were obtained from the in-house breeding system (details maintained within the centrally held records). Viable fish eggs were obtained from breeding groups of Pimephales promelas. Prior to initiation of the test the stage of development was established. The eggs were added to the test system, typically before the first cleavage of the blastodisc or within 24 hours of fertilisation.
The breeding groups in the breeding system were held in a temperature-controlled room under artificial light (with a 16 hour light:8 hour dark photo-period with a ca 30 minute dawn/dusk period) in holding tanks appropriate to their size, under continuous water renewal (flow-through) conditions.
The breeding groups of Pimephales promelas were fed ad libitum using freshly hatched brine shrimp (Artemia salinis) nauplii and also with Tetramin® flake food, which was added to the holding tank in quantities dictated by the size of the fish. The food was not considered to contain contaminants likely to affect the outcome of the study. Uneaten food and debris was siphoned or cleaned from the tanks as required.
The water used was laboratory treated mains supply. The water was pumped to the laboratory through an activated carbon filter.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

32 d

Hardness:

16.0 mg/l

Test temperature:

15 deg C

pH:

7.3

Dissolved oxygen:

Control: 94 – 97
Solvent control: 95 – 96
0.80: 95 – 97
1.6: 96 – 97
8.0: 94 – 96
16: 95
80: 95 – 96

Salinity:

Not applicable.

Conductivity:

180 uS/cm

Nominal and measured concentrations:

Nominal test substance concentrations of 0.80, 2.6, 8.0, 26 and 80 µg/L.

Details on test conditions:

Based on the results of the range-finding test, the definitive test was conducted at nominal test substance concentrations of 0.80, 2.6, 8.0, 26 and 80 µg/L. Control and solvent control groups were also included.
The test was conducted using a flow-through design (continual renewal of the media).
A solvent stock solution of 1.6 mg/mL was prepared by dissolving ca 400 mg of test substance in 250 mL of dimethylformamide (DMF). Serial dilutions were prepared in DMF to give further solvent stock solutions of 0.52, 0.16, 0.052 and 0.016 mg/mL.
The 0.016, 0.052, 0.16, 0.52 and 1.6 mg/mL solvent stock solutions were each separately dosed at a rate of 0.06 mL/hour to a diluent flow rate of 20 ± 10% mL/min to give the 0.80, 2.6, 8.0, 2.6 and 80 µg/L test concentrations, respectively.
A solvent control was prepared in a similar manner by dosing solvent only at a rate of 0.06 mL/hour to a diluent flow rate of 20 ± 10% mL/min.
Four replicates were prepared for the solvent control and each test concentration.
A dilution water control was prepared by continuously delivering treated mains water only into the four control vessels.
Solvent stock solution usage and diluent flow rates were checked daily during the test.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

> 80 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

26 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Post hatch survival

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

26 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Statistical analysis of the data was performed using the CETIS program v 1.8.6.8.

Hatching success and post-hatch survival are considered to be quantal data. Lengths and weights are considered to be continuous data.

The data (hatching success, post-hatch survival, lengths and weights) were shown to be normally distributed with equal variance.

The control and solvent control groups were compared using the Fisher Exact Test (hatching success and post-hatch survival) and Equal Variance t Two sample Test (lengths and weights).

No significant differences were observed in terms of post-hatch survival, lengths and weights however a significant difference was observed between the control and solvent control groups in terms of hatching success. As such, the test groups were compared to the solvent control group.

The hatching success data were analysed using the Dunnett Multiple Comparison Test. No significant differences were observed between the solvent control and test groups.

The post-hatch survival data were analysed using the Cochran-Armitage Trend Step-Down Test. A statistical outlier was observed in the 80 µg/L test group (Grubbs Extreme Value Test). Data were therefore analysed with and without the outlying replicate. Both analyses showed a significant difference between the solvent control and 80 µg/L test group.

The length and weight data were analysed using the Dunnett Multiple Comparison Test. No significant differences were observed between the solvent control and test groups in terms of wet weight. A statistical outlier replicate was observed in the 26 µg/L test group. Statistical analysis including all replicates showed a statistically significant difference between the 26 µg/L test group and the solvent control with no significant differences between the solvent control and remaining treatment groups including the 80 µg/L test group. Statistical analysis excluding the outlier replicate showed a significant difference between the solvent control and 80 µg/L test group. To confirm this result, the data from the solvent control and 80 µg/L only were analysed using the Equal Variance t Two sample Test. The result from this analysis showed a statistically significant difference between the solvent control and 80 µg/L test group.

Results with reference substance (positive control):

Not applicable.

 Biological Data

Nominal concentration (µg/L)	Hatching success (%)	Day 28 Post-hatch survival (%)	Mean total length (mm)	Mean total weight (mg)
Control	99	94	22.5	98.6
Solvent control	86	93	22.6	103.9
0.80	89	95	22.2	101.5
2.6	98	92	22.0	101.2
8.0	100	91	21.4	93.7
26	98	88	20.2	82.9
80	91	62	21.2	100.4

 

Chemical analysis

 

	Measured concentration (µg/L)
Nominal concentration (µg/L)	Day 0	Day 4 (pre-hatch)	Day 4 (post-hatch)	Day 10 (post-hatch)
Control	-	-	-	-
Solvent control	-	-	-	-
0.80	0.224	0.290	0.380	0.985
2.6	0.626	0.838	0.672	2.09
8.0	0.767	2.03	3.00	7.89
26	7.81	4.56	3.46	17.0
80	14.4	14.5	21.6	22.2

– non detected above LOQ (0.2 µg/L)

	Measured concentration (µg/L)
Nominal concentration (µg/L)	Day 18 (post-hatch)	Day 24 (post-hatch)	Day 28 (post-hatch)	GMM (µg/L)
Control	-	-	-	-
Solvent control	-	-	-	-
0.80	0.730	0.667	0.817	0.50
2.6	2.22	1.45	2.08	1.3
8.0	4.20	4.27	12.9	3.6
26	14.4	11.3	23.3	9.7
80	26.5	20.0	49.0	22

– non detected above LOQ (0.2 µg/L)
GMM = geometric mean measured concentration

Validity criteria fulfilled:

yes

Conclusions:

No significant differences were observed in terms of post-hatch survival, lengths and weights however a significant difference was observed between the control and solvent control groups in terms of hatching success. As such, the test groups were compared to the solvent control group.

It should be noted that the water solubility of the test substance at 20°C is < 4 μg/l. The effects noted in this study are above the limit of solubility of the test substance in water, and are based on statistical manipulation. It is considered that these effects are attributable to solvent effects and the magnification of the effects is caused by the action of the solvents rather than any effects from the test substance which would not be found at these levels within the environment. As such, the effect concentrations are considered to be subject to variability due to the low solubility in water and solvent and as such, the test substance is proposed to not be harmful at the limit of solubility in water to fish or aquatic organisms in general.

Executive summary:

No significant differences were observed in terms of post-hatch survival, lengths and weights however a significant difference was observed between the control and solvent control groups in terms of hatching success. As such, the test groups were compared to the solvent control group.

It should be noted that the water solubility of the test substance at 20°C is < 4 μg/l. The effects noted in this study are above the limit of solubility of the test substance in water, and are based on statistical manipulation.  It is considered that these effects are attributable to solvent effects and the magnification of the effects is caused by the action of the solvents rather than any effects from the test substance which would not be found at these levels within the environment. As such, the effect concentrations are considered to be subject to variability due to the low solubility in water and solvent and as such, the test substance is proposed to not be harmful at the limit of solubility in water to fish or aquatic organisms in general.  

Description of key information

The NOEC is greater than the water solubility for the substance.

Additional information

No significant differences were observed in terms of post-hatch survival, lengths and weights however a significant difference was observed between the control and solvent control groups in terms of hatching success. As such, the test groups were compared to the solvent control group.

It should be noted that the water solubility of the test substance at 20°C is < 4 μg/l. The effects noted in this study are above the limit of solubility of the test substance in water, and are based on statistical manipulation.  It is considered that these effects are attributable to solvent effects and the magnification of the effects is caused by the action of the solvents rather than any effects from the test substance which would not be found at these levels within the environment. As such, the effect concentrations are considered to be subject to variability due to the low solubility in water and solvent and as such, the test substance is proposed to not be harmful at the limit of solubility in water to fish or aquatic organisms in general.