6-tert-butyl-2,4-xylenol

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:

29 October 2021 to 09 December 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD (2013). Guidelines for the Testing of Chemicals. TG 210: Fish, Early Life-Stage Toxicity Test. OECD, Paris.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Study samples were extracted into 1 mg/L naphthalene (internal standard) in hexane, diluted to within the calibration range with 1 mg/L naphthalene (internal standard) in hexane, if required, and analysed against standard solutions of 6-tert-butyl-2,4-xylenol of known purity.

Vehicle:

no

Remarks:

This study was run with a dilution water control, solvent control and nominal 6-tert-butyl-2,4-xylenol concentrations

Details on test solutions:

This study was run with a dilution water control, solvent control and nominal 6-tert-butyl-2,4-xylenol concentrations of 0.01, 0.03, 0.1, 0.32 and 1.0 mg/L.
Dosing stocks were prepared by dissolving a known amount of 6-tert-butyl-2,4-xylenol (see table below) in 50 mL of TEG in a volumetric flask. A stock was prepared consisting of only TEG in a volumetric flask for the solvent control. All stocks were treated similarly, with shaking and 20 minutes ultrasonication used to dissolve the test substance into solution. All stocks were observed to be clear and colourless prior to transfer to the syringe dosing apparatus.
The syringe pump delivered the TEG stocks into the mixing chambers at a nominal rate of 4 μL/min. This was mixed with the incoming dilution water at a nominal 400 mL/min. This dilution of the TEG stock in the dilution water delivered the nominal test substance concentration. The dilution water control contained dilution water only with no solvent addition. The solvent control and the and nominal concentrations 0.01, 0.03, 0.1, 0.32 and 1.0 mg/L contained the same nominal TEG concentration of 10 μL/L.
The test solutions (the water in the aquaria) were observed to be clear and colourless on Days -6, 0, 5 (hatch day),12, 19, 26, and 33. The mixing cells were covered with aluminium foil at the start of the test in order to minimise microbial growth.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The test organism was the fathead minnow (P. promelas). This species is readily available and easily bred in the laboratory. The species is representative of a widely distributed freshwater fish and is a recommended species by the OECD 210 TG.
The eggs required to start the test were obtained from broodstock adult fish held at Scymaris. The broodstock were held under similar conditions as those used during the study. The broodstock were held under artificial lighting and fed daily using a commercial fish food and frozen adult brine shrimp (Artemia spp). Broodstock showed no evidence of disease during the two months prior to study.

Feeding regime
The hatched larvae/fry were fed twice daily (except for the start and end of the exposure). On Day 4 (the day prior to hatch day), a very small amount of powdered commercial food was added to each egg cup to avoid any starvation effects in the hatched fry. From hatch day until Day 7 post-hatch, the fry were fed on a powdered commercial fry food fed ad libitum per tank per feed. Newly hatched brine shrimp Artemia (typically up to 60 mL of culture per tank per feed) were fed from Day 2 post-hatch. A pelleted commercial food, fed ad libitum from Day 20 post-hatch, was also introduced into the diet. Feeding was in excess, while not reducing water quality conditions, and approximately equal across replicates based on numbers of fry.
Representative batches of the food types used in this study were analysed for pesticides, PCBs and metals. These non-study background analyses were conducted under non-GLP conditions by Fera Science Ltd, York

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Remarks on exposure duration:

egg to 28 days post-hatch

Post exposure observation period:

No post exposure observation period specified

Hardness:

85 CaCO3 (mg/L)

Test temperature:

The temperature of the test solutions measured on six occasions throughout the study ranged from 24.6 to 25.9°C.

pH:

The overall minimum and maximum pH values were 7.24 and 7.85. The overall mean pH value was 7.53.

Dissolved oxygen:

The overall minimum and maximum DO concentrations were 87.8 and 99.0% ASV. The overall mean DO concentration was 91.9% ASV.

Salinity:

Not specified

Conductivity:

264 μS/cm

Nominal and measured concentrations:

nominal concentrations of 0.01, 0.03, 0.1, 0.32 and 1.0 mg/L.

Details on test conditions:

Test apparatus
The test was undertaken in a temperature-controlled room which was set at the nominal test temperature of 25 ± 1.5°C. The test vessels were glass aquaria of approximately 9.5 litre working capacity with four replicate aquaria per treatment. A flow-through test system was used for this study.
Egg incubation cups (1 per replicate tank) made from 8 cm lengths of 5 cm diameter glass tubing with nylon mesh stuck to the bottom of each cup using silicone sealant were used. The cups were suspended in the test vessels and oscillated vertically approximately 20-50 mm at 2 oscillations per minute to maintain a non-turbulent water flow over the eggs while ensuring complete submergence of the eggs.
The dilution water was fed from a temperature-controlled header tank via a flow control device to mixing chambers at a nominal rate of 400 mL/min. All chambers, with the exception of the controls, also received the required amount of 6-tert-butyl-2,4-xylenol dissolved into TEG (tri-ethylene glycol) and supplied by syringe pump at a rate of 4 μL/min. The solvent control chamber received just TEG at the required rate to give the same solvent concentration as in the test concentrations (except the control). Separate lines from each chamber supplied at least 5 tank volumes replacements per day to each of the 4 replicate test vessels.
Measurement of the dilution water delivery systems and the incoming flow rate for each aquarium was monitored by direct measurement of flow rates. The syringe pump dosing was confirmed on Day -6, 0, 5, 12, 19 and 26 over a known time interval. Measurements of the dilution water delivery and aquaria inflow rates were made on Day -6, 0 and at hatch (Day 5), then at least weekly for the duration of the test, with daily visual checks to ensure the correct operation of the entire dosing system. Additional measurements and adjustments of the dilution water delivery system were made on Day -2. The dilution water flow rates were maintained within ±10% of nominal. The aquaria flow rates were checked weekly and generally maintained within ±20% throughout the study, both within each aquarium and between the aquaria for the duration of the test. The only exceptions were during the pre-exposure dosing phase (±29%), at the end of the test (28 days post-hatch) where the maximum measured flow rate was 126% of the minimum flow rate, and in the nominal 0.01 mg/L replicate C test vessel where the maximum measured flow rate over the duration of the exposure was 123% of the minimum flow rate. These relatively minor deviations from the required range are not thought to have had any effect on the outcome of the test or the welfare of the fish.
The fish stocking density did not exceed 0.5 g/L/day or 5 g/L at any time, this was confirmed using wet weight data at test end.
The position of the treatments followed a randomised block design. Each treatment was within a block position in the experimental area randomised from left to right.

Test procedure
The dosing of 6-tert-butyl-2,4-xylenol was started on 29 October 2021. The exposure commenced on 04 November 2021 by placing a nominal 20 embryos, <24 hours old, into the incubation cup in each replicate test tank (nominal 80 embryos in each control group and nominal concentrations 0.01, 0.03, 0.1, 0.32 and 1.0 mg/L). Embryos from 12 breeding groups (1 group: 4 males and 8 females) were used.
The number of live and dead embryos were recorded daily, with dead embryos being discarded. When hatching was ≥ 90% complete in the dilution water control all hatched fry were released into the test vessels. Daily observations of fry mortality, behaviour and appearance were made and recorded throughout the study. Test vessels were cleaned, when necessary, throughout the study.
The study was terminated on Day 28 post-hatch. At the end of the exposure period, the survival, total length, and wet weight (blotted dry) were recorded along with the total dry weight of the population. Any physical abnormalities were also recorded.
The photoperiod used was 16 hours of light: 8 hours of dark, with 20-minute dawn: dusk transition periods.

Physical and Chemical parameters
The test solution pH, dissolved oxygen (DO) and temperature was measured in all test vessels at test start (embryo exposure), hatch day, then subsequently once a week for the duration of the study. The temperature of replicate D of the dilution water control was monitored continuously for the duration of the study by a min/max thermometer.
The water hardness was measured in replicate A of the dilution water control and nominal concentration 1.0 mg/L on exposure Day 0. The water hardness was not measured on Day 28 post-hatch in error, however the water hardness of the laboratory freshwater supply, measured four days prior to the end of the test, is provided. As the hardness results were consistent between the start of the test and the freshwater supply close to the end of the test, it is not thought that this deviation caused any impact on the outcome of the study.
The light intensity at aquaria water level in approximately the middle of the test rig was measured on a single occasion during the test. The light intensity was reduced as much as possible in order to minimise microbial growth in the test system.

Biological analysis
All biological analysis was performed using the statistical package ToxRat.
The effect concentration (ECx) is defined as the concentration of the test substance, calculated from the data obtained, predicted to result in an x% adverse effect within the stated exposure period. Where no responses were observed, the ECx values was reported as greater than the highest test concentration.
The LOEC, the lowest concentration of 6-tert-butyl-2,4-xylenol observed to have a significant effect (p <0.05) and the NOEC, the concentration directly below the LOEC, were determined using statistical analysis and sound scientific judgement to determine if they were biologically meaningful.

Hatching and survival post-hatch
Replicate data were pooled and the dilution water control and solvent control data for hatch and survival post-hatch were compared by Fisher’s Exact Binomial Test. This test was chosen in place of a t-test or Mann-Whitney test to avoid unnecessary transformation of the data and is recommended by OECD statistical guidance. The dilution water control and solvent control were then pooled and compared to the 6-tert-butyl-2,4-xylenol treatment concentrations for hatch and survival post-hatch data using Dunnett’s Multiple t-test (after arcsine transformation). Transformation of each set of data was required to allow use of parametric testing which gave a greater statistical power.

Length and weight
Comparisons of total length, wet weight and dry weight were made between the dilution water control and the solvent control using a Student t-test, where no significant effect was found the controls were pooled for further comparison to the test groups. Where a significant effect was found, the solvent control was used for comparison to the test groups. The mean total length, wet weight and dry weight data from each replicate test vessel (individual fry data pooled) were assessed for normality and equality of variance. The statistical test used in comparison of the pooled or solvent control to the 6-tert-butyl-2,4-xylenol treatment concentrations for total length, wet weight and dry weight was the Williams Multiple Sequential t-test.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Details on results:

Behaviour and appearance
On Day 5 (hatch day), five fry (one in each of the dilution water control and solvent control, two in the nominal 0.01 mg/L concentration and one in the nominal 0.32 mg/L concentration) were found to be malformed and unable to swim or feed, these were euthanised at that time for ethical reasons. Similarly, on Day 5 (hatch day), one fry in the solvent control was recorded as malformed but healthy and three fry (one in the nominal 0.01 mg/L concentration and two in the nominal 0.1 mg/L concentration) were observed to have bent spines.
On Day 15 (10 days post-hatch), five fry (one from the nominal 0.03 mg/L concentration and four from the nominal 1.0 mg/L concentration) were euthanised due to bent spines which were thought to be causing suffering. On Day 21 (16 days post-hatch), two fry from the nominal 1.0 mg/L concentration were euthanised to minimise suffering. One was observed as small and continually swimming in a corkscrew motion, the other fry was observed to have a loss of equilibrium (swimming upside down) and hypoactivity.
One further fry, from the solvent control, was euthanised on Day 26 (21 days post-hatch) due to a severely bent spine, this fry was also observed to be smaller than the other fry in the same test vessel.
At the end of the test (28 days post-hatch), the following numbers of fry were observed to have a bent spine. These were not euthanised during the exposure as they otherwise appeared healthy.
The relatively high number of fry with bent spines during this study was thought to possibly due to poor quality eggs from one or more of the parent adult female fish. As the eggs from the different parents were mixed before distribution to the test vessels, this would explain why the malformation appeared to be prevalent across the concentration range.

Control comparison
No significant differences (p <0.05) were found between the dilution water control and the solvent control groups for the hatch, survival, total length or dry weight endpoints; therefore, the controls were pooled and statistical comparisons were carried out between the pooled controls and 6-tert-butyl-2,4-xylenol treatment groups.
A significant difference (p <0.05) was found between the dilution water control and solvent control groups for the wet weight endpoint. In this case, statistical comparisons were carried out between the solvent control and 6-tert-butyl-2,4-xylenol treatment groups. This was not thought to be due to an effect of the solvent as the nominal 0.03 and 0.1 mg/L treatments had mean wet weights which were either similar to, or greater than, the dilution water control mean wet weight. As the solvent concentration in these treatments was the same as the solvent control, this indicated there was no solvent effect on wet weight. However, to avoid any doubt in this regard, statistical comparisons were made to the solvent control.

Hatch data
Hatch day in this study was 09 November 2021, which was exposure Day 5.
The range of percentage hatch success across all replicates was 81 to 100%. The mean percentage hatch success across all replicates in the dilution water control and solvent control were 95 and 98%, respectively.
A linear max. likelihood regression was conducted on the pooled control and treatment hatch data, however the ECx values could not be determined due to the lack of a concentration response. In this case, the ECx values have been reported as >1.0 mg/L.
For two replicates on Day 2 (solvent control replicate A and 0.01 mg/L replicate C), the number of eggs recorded increased by one between the start and end of the daily observations with no further information. In both cases, based on the number of dead eggs removed and number of hatched fish, it was assumed that 21 eggs (rather than the nominal 20 eggs) had been added to these replicates at the start of the test in error.
On Day 4, only 18 total eggs/fry were observed in solvent control replicate D. During observations, fry were seen swimming freely through the mesh in the egg cup. It was assumed that the two missing fry had hatched and escaped into the tank. The egg cup was replaced to prevent any further escapes.

Survival data
The range of percentage survival post-hatch across all replicates was 65 to 100%. The mean percentage survival post-hatch in the dilution water control and solvent control were 88 and 89%, respectively.
A linear max. likelihood regression was conducted on the pooled control and treatment survival data, however the ECx values could not be determined due to the lack of a concentration response. In this case, the ECx values have been reported as >1.0 mg/L.
On Day 4, only 18 eggs/fry were observed in solvent control replicate D. During observations, fry were seen swimming freely through the mesh in the egg cup. It was assumed that the two hatched fry had escaped and died during the exposure, based on the number of surviving fry at the end of the test.

Length data
The total lengths of the fry in the dilution water control ranged from 17.50 to 28.01 mm with a mean of 23.28 mm. The total lengths of the fry in the solvent control ranged from 14.08 to 28.20 mm with a mean of 22.32 mm. The mean length of the dilution water control and solvent control both exceeded the 18 mm typical control minimum mean total length requirement of the OECD 210 TG.
A non-linear regression was conducted on the pooled control and treatment total length data to determine the ECx values.

Weight data
The wet weights of the fry in the dilution water control ranged from 0.04185 to 0.23055 g with a mean of 0.11712 g. The wet weights of the fry in the solvent control ranged from 0.01845 to 0.19129 g with a mean of 0.0.10332 g.
A non-linear regression was conducted on the solvent control and treatment wet weight data to determine the ECx values.

It was noticed after the procedures at the end of the test, that one of the wet weights of the fry in 0.1 mg/L replicate D had not been recorded in error. There were 18 individual fry length values recorded for this replicate but only 17 individual fry wet weight values. The calculated means were based on the number of fry recorded for each endpoint. As the mean wet weights for each replicate were used in the statistical comparisons, and 17 of the 18 fry were recorded successfully, the effect of this deviation on the results used for endpoint determinations was thought to be very minor.
The calculated dry weights of individual fry (estimated using the dry weight of the pooled fry from each replicate) in the dilution water control ranged from 0.02306 to 0.02681 g with a mean of 0.02476 g. The calculated dry weights of individual fry in the solvent control ranged from 0.01967 to 0.02353 g with a mean of 0.02205 g.
A non-linear regression was conducted on the pooled control and treatment dry weight data to determine the ECx values.

Overall NOEC
Including all of the assessed endpoints at the end of the study, and using the nominal concentrations, the overall LOEC for 6-tert-butyl-2,4-xylenol in this study was 1.0 mg/L, and the overall NOEC was 0.32 mg/L.

Physical and chemical data
The pH values of the test solutions, measured throughout the study ranged from 7.24 to 7.85. pH. The overall minimum and maximum pH values were 7.24 and 7.85. The overall mean pH value was 7.53.
The dissolved oxygen (DO) concentrations of the test solutions, measured throughout the study ranged from 87.8 to 99.0% of the air saturation value (ASV). The overall minimum and maximum DO concentrations were 87.8 and 99.0% ASV. The overall mean DO concentration was 91.9% ASV.
The water hardness measured in the dilution water control and nominal 1.0 mg/L treatment test solutions at test start were 80.7 and 83.0 mg/L CaCO3, respectively. The water hardness in the test solutions was not measured at the end of the test in error, however the water hardness of the laboratory freshwater supply (used as the dilution water control and as part of the test solutions) was measured four days prior to the end of the test and found to be 81.6 mg/L CaCO3.
The temperature of the test solutions measured on six occasions throughout the study ranged from 24.6 to 25.9°C.
The continuous temperature measurement, recorded in the dilution water control replicate D test vessel, ranged from 25.0 to 25.9°C.
Lux was measured at the start of the test at the aquaria water level in approximately the middle of the test rig and was determined to be 155 lux (cosine).
Representative batches of the food used in this study were analysed for pesticides, PCBs and metals.

Aquaria flow rates
The overall range of the aquaria flow rates, measured for each test aquaria throughout the exposure ranged from 86 to 110 mL/min and had an overall mean of 101 mL/min. This mean value provides a nominal 15.3 tank volume (9.5 litre) replacements in 24 hours.

Stocking density
The maximum stocking density for individual aquaria was calculated at test end by using the wet weights of the fry recovered, the nominal tank volume and the mean flow rate recorded for each test vessel. The stocking density in each test vessel at test end was below the maximum permitted 5 g/L wet weight at any time (Ref 1), with a range of 0.12 – 0.23 g/L wet weight. It was also below the maximum permitted value of 0.5 g/L/day, with a range of 0.010 – 0.015 g/L/day.

Reported statistics and error estimates:

Hatch data
No significant difference (p <0.05) was found between the control groups (Fisher’s Exact Binomial test). No significant differences (p <0.05) were found between the pooled controls and the treatment concentrations for hatch data (Dunnett’s Multiple t-test performed after arcsine transformation of the data).

Survival data
No significant difference (p <0.05) was found between the control groups (Fisher’s Exact Binomial test). No significant differences (p > 0.05) were found between the pooled controls and the treatment concentrations (Dunnett’s Multiple t-test performed after arcsine transformation of the data).

Length data
No significant difference (p <0.05) was found between the control groups (Student t-test). No significant differences (p <0.05) were found between the length of the pooled controls and the nominal 0.01, 0.03, 0.1 or 0.32 mg/L test concentrations, a significant difference (p <0.05) was found for the nominal 1.0 mg/L concentration (Williams Multiple Sequential t-test).

Weight data
A significant difference (p <0.05) was found between the dilution water control and the solvent control groups (Student t-test; Section 5.2.2). No significant differences (p <0.05) were found between the wet weight in the solvent control and the nominal 0.01, 0.03, 0.1 and 0.32 mg/L test concentrations, a significant difference (p <0.05) was found for the nominal 1.0 mg/L concentration (Williams Multiple Sequential t-test).
No significant differences (p <0.05) were found between the dry weight of the pooled controls and the nominal 0.01, 0.03, 0.1 and 0.32 mg/L test concentrations, a significant difference (p <0.05) was found for the nominal 1.0 mg/L concentration (Williams Multiple Sequential t-test).

Hatch and Survival Data

Nominal concentration of 6-tert-butyl-2,4-xylenol (mg/L)	Replicate	Eggs introduced Day 0	Total number hatched	% hatched		Number of fry surviving	% survival post-hatch
				Replicate	Treatment		Replicate	Treatment
Dilution water control	A	20	17	85	95	14	82	88
	B	20	20	100		18	90
	C	20	19	95		17	89
	D	20	20	100		18	90
Solvent control	A	21	20	95	98	16	80	89
	B	20	20	100		20	100
	C	20	19	95		16	84
	D	20	20	100		18	90
0.01	A	20	17	85	89	14	82	82
	B	20	19	95		17	89
	C	20	17	85		12	71
	D	20	18	90		15	83
0.03	A	20	19	95	96	15	79	82
	B	20	19	95		15	79
	C	20	19	95		15	79
	D	20	20	100		18	90
0.1	A	20	20	100	94	13	65	83
	B	20	17	85		15	88
	C	20	19	95		16	84
	D	20	19	95		18	95
0.32	A	20	18	90	94	16	89	93
	B	20	17	85		17	100
	C	20	20	100		17	85
	D	20	20	100		20	100
1.0	A	20	20	100	93	15	75	80
	B	20	18	90		12	67
	C	21	17	81		17	100
	D	20	20	100		16	80

No statistically significant (p >0.05) differences between the pooled controls and the treatment groups were observed for the Hatch or Survival post-hatch endpoints.

Percentages are quoted to the nearest integer.

Solvent control replicate A and 1.0 mg/L replicate C: 21 eggs added at test start inadvertently.

 

Summary total length data

Nominal concentration of 6-tert-butyl-2,4-xylenol (mg/L)	Replicate	Number of fry	Mean total length (mm)	Minimum total length (mm)	Maximum total length (mm)	Standard deviation (mm)
Dilution water control	A	14	22.7	17.50	25.51	2.34
	B	18	23.59	19.05	26.39	1.98
	C	17	23.96	21.10	28.01	2.04
	D	18	22.74	20.77	25.22	1.17
	Pooled	67	23.28	17.50	28.01	1.93
Solvent control	A	16	22.75	18.83	25.39	2.21
	B	20	21.69	15.82	25.96	2.49
	C	16	22.56	14.08	28.20	3.25
	D	18	22.42	17.06	25.08	2.15
	Pooled	70	22.32	14.08	28.20	2.53
0.01	A	14	23.32	21.31	26.04	1.55
	B	17	22.40	18.42	25.52	1.90
	C	12	23.16	17.43	26.72	2.91
	D	15	21.98	12.62	25.64	3.28
	Pooled	58	22.67	12.62	26.72	2.48
0.03	A	15	23.74	18.16	26.56	2.61
	B	15	22.94	16.38	27.17	3.08
	C	15	22.93	19.96	25.82	1.61
	D	18	23.29	16.50	25.50	2.28
	Pooled	63	23.23	16.38	27.17	2.41
0.1	A	13	23.72	15.18	27.15	3.61
	B	15	23.31	18.51	25.83	1.83
	C	16	23.71	19.80	26.02	1.77
	D	18	22.68	18.24	25.50	2.01
	Pooled	62	23.32	15.18	27.15	2.33
0.32	A	16	22.31	16.80	25.15	2.28
	B	17	22.59	19.92	25.62	1.56
	C	17	22.33	11.59	25.66	3.26
	D	20	22.14	14.37	25.92	2.43
	Pooled	70	22.33	11.59	25.92	2.41
1.0	A	15	21.96	18.44	26.49	1.91
	B	12	21.04	13.12	24.67	3.30
	C	17	20.87	13.87	23.54	2.29
	D	16	20.71	16.83	25.13	2.55
	Pooled	60	21.13	13.12	26.49	2.49

No statistically significant (p <0.05) differences between the pooled controls and the treatment groups were observed for the total length endpoint.

Lengths reported to 2 decimal places. Pooled means calculated using individual fry length values.

 

Summary wet weight data

Nominal concentration of 6-tert-butyl-2,4-xylenol (mg/L)	Replicate	Number of fry	Mean wet weight (g)	Minimum wet weight (g)	Maximum wet weight (g)	Standard deviation (g)
Dilution water control	A	14	0.11180	0.04185	0.16807	0.03714
	B	18	0.12086	0.06259	0.18167	0.02989
	C	17	0.12531	0.07750	0.23055	0.03825
	D	18	0.10980	0.06722	0.16181	0.02170
	Pooled	67	0.11712	0.04185	0.23055	0.03193
Solvent control	A	16	0.11034	0.05222	0.15407	0.03335
	B	20	0.09445	0.03554	0.15644	0.03302
	C	16	0.10831	0.01845	0.19129	0.04236
	D	18	0.10248	0.04395	0.13971	0.02682
	Pooled	70	0.10332	0.01845	0.19129	0.03391
0.01	A	14	0.11891	0.08079	0.16086	0.02372
	B	17	0.09877	0.05156	0.12850	0.02086
	C	12	0.12060	0.04670	0.18483	0.04697
	D	15	0.10360	0.01464	0.15634	0.03822
	Pooled	58	0.10940	0.01464	0.18483	0.03356
0.03	A	15	0.13011	0.05984	0.17841	0.03929
	B	15	0.11164	0.04138	0.20142	0.04665
	C	15	0.10689	0.06809	0.16099	0.02822
	D	18	0.11541	0.03260	0.15523	0.03270
	Pooled	63	0.11598	0.03260	0.20142	0.03723
0.1	A	13	0.13276	0.04870	0.20314	0.04825
	B	15	0.11609	0.06007	0.16944	0.02989
	C	16	0.13085	0.080878	0.17429	0.03036
	D	17	0.10661	0.04728	0.14887	0.02733
	Pooled	61	0.12087	0.04728	0.20314	0.03499
0.32	A	16	0.1034	0.04687	0.13080	0.02272
	B	17	0.09924	0.05628	0.14776	0.02554
	C	17	0.10663	0.01026	0.16290	0.03763
	D	20	0.09718	0.02173	0.14483	0.03045
	Pooled	70	0.10136	0.01026	0.16290	0.02934
1.0	A	15	0.09781	0.04896	0.18683	0.03288
	B	12	0.09240	0.02200	0.16160	0.03933
	C	17	0.08437	0.02281	0.11126	0.02418
	D	16	0.08217	0.03314	0.16300	0.03416
	Pooled	60	0.08875	0.02200	0.18683	0.03226

No statistically significant (p <0.05) differences between the solvent control and the treatment groups listed above observed for the wet weight endpoint.

Wet weights reported to 5 decimal places. Pooled means calculated using individual fry wet weight values.

 

Dry weight data

Nominal concentration of 6-tert-butyl-2,4-xylenol (mg/L)	Replicate	Mean dry weight per fry (g)	Treatment mean dry weight per fry (g)	Standard deviation (g)
Dilution water control	A	0.02401	0.02476	0.00162
	B	0.02518
	C	0.02681
	D	0.02306
Solvent control	A	0.02331	0.02205	0.00179
	B	0.01967
	C	0.02353
	D	0.02170
0.01	A	0.02420	0.02285	0.00161
	B	0.02103
	C	0.02421
	D	0.02196
0.03	A	0.02754	0.02470	0.00208
	B	0.02361
	C	0.02277
	D	0.02488
0.1	A	0.02898	0.02617	0.00263
	B	0.02465
	C	0.02774
	D	0.02332
0.32	A	0.02123	0.02156	0.00096
	B	0.02124
	C	0.02296
	D	0.02079
1.0*	A	0.02034	0.01930	0.00131
	B	0.02038
	C	0.01767
	D	0.01880

*Statistically significant (p <0.05) difference when compared to the pooled controls.

 

Test vessels pH values

Nominal concentration of 6-tert-butyl-2,4-xylenol (mg/L)	Replicate	pH
		Day 0	Hatch Day	Day 7 post-hatch	Day 14 post-hatch	Day 21 post-hatch	Day 28 post-hatch
Dilution water control	A	7.32	7.66	7.24	7.35	7.44	7.29
	B	7.38	7.69	7.25	7.39	7.43	7.34
	C	7.45	7.72	7.26	7.44	7.46	7.35
	D	7.48	7.74	7.26	7.46	7.52	7.36
Solvent control	A	7.52	7.46	7.27	7.46	7.52	7.36
	B	7.53	7.36	7.27	7.47	7.53	7.37
	C	7.55	7.60	7.29	7.48	7.54	7.37
	D	7.58	7.63	7.29	7.49	7.57	7.39
0.01	A	7.59	7.74	7.28	7.49	7.58	7.40
	B	7.61	7.75	7.27	7.49	7.57	7.40
	C	7.62	7.76	7.26	7.48	7.58	7.40
	D	7.64	7.77	7.27	7.50	7.58	7.41
0.03	A	7.71	7.78	7.32	7.51	7.59	7.41
	B	7.72	7.82	7.33	7.51	7.59	7.41
	C	7.71	7.81	7.33	7.52	7.60	7.43
	D	7.71	7.80	7.31	7.52	7.60	7.42
0.1	A	7.75	7.81	7.38	7.56	7.59	7.42
	B	7.76	7.80	7.37	7.56	7.60	7.42
	C	7.76	7.82	7.36	7.55	7.61	7.43
	D	7.71	7.82	7.34	7.55	7.61	7.43
0.32	A	7.67	7.83	7.28	7.54	7.62	7.44
	B	7.68	7.83	7.29	7.55	7.62	7.45
	C	7.71	7.84	7.31	7.56	7.63	7.46
	D	7.73	7.84	7.30	7.56	7.63	7.47
1.0	A	7.76	7.85	7.32	7.57	7.65	7.47
	B	7.76	7.84	7.35	7.58	7.66	7.48
	C	7.76	7.83	7.34	7.59	7.65	7.48
	D	7.76	7.84	7.32	7.59	7.65	7.48

All measurements are quoted to 2 decimal places

 

Test vessels dissolved oxygen (DO) concentrations

Nominal concentration of 6-tert-butyl-2,4-xylenol (mg/L)	Replicate	DO % Air Saturation Value (ASV)
		Day 0	Hatch Day	Day 7 post-hatch	Day 14 post-hatch	Day 21 post-hatch	Day 28 post-hatch
Dilution water control	A	93.5	95.5	93.8	93.9	91.9	93.3
	B	94.1	95.1	93.2	91.5	91.0	92.9
	C	90.5	95.3	92.8	91.2	91.8	92.1
	D	90.5	95.0	99.0	92.3	91.3	90.4
Solvent control	A	92.1	95.6	93.9	93.4	91.6	90.2
	B	90.8	94.5	93.7	92.1	91.3	89.5
	C	93.1	96.3	93.9	92.0	90.7	89.4
	D	93.2	95.2	92.7	91.5	91.3	89.4
0.01	A	92.1	95.6	92.1	91.8	90.8	89.0
	B	93.0	95.1	92.4	90.9	90.0	89.7
	C	93.0	95.8	92.5	91.6	90.5	90.2
	D	93.9	95.7	91.6	91.6	91.3	90.9
0.03	A	91.9	94.1	92.1	90.1	89.9	88.4
	B	93.0	94.1	91.3	90.8	90.3	90.7
	C	93.0	95.8	92.5	90.4	90.4	89.6
	D	94.3	95.2	92.1	90.1	89.7	88.8
0.1	A	92.7	95.2	91.8	90.5	90.7	87.7
	B	92.2	94.8	92.3	91.7	90.9	88.1
	C	90.6	94.7	90.5	90.9	89.2	88.6
	D	92.5	95.4	91.54	91.3	89.4	89.0
0.32	A	93.0	94.7	91.3	90.8	90.5	88.7
	B	93.6	94.8	91.5	91.0	90.4	90.0
	C	94.1	95.2	93.0	91.6	90.0	88.5
	D	93.0	94.5	92.0	89.6	89.7	89.3
1.0	A	91.3	94.2	91.8	89.8	89.2	88.2
	B	92.3	94.1	92.1	90.4	89.3	89.1
	C	91.6	93.9	92.7	90.9	91.1	88.6
	D	92.0	94.3	91.7	90.6	88.6	88.6

All measurements are quoted to 1 decimal place

 

Test vessel temperature data

Nominal concentration of 6-tert-butyl-2,4-xylenol (mg/L)	Replicate	pH
		Day 0	Hatch Day	Day 7 post-hatch	Day 14 post-hatch	Day 21 post-hatch	Day 28 post-hatch
Dilution water control	A	25.1	25.5	25.7	25.5	25.6	25.4
	B	25.1	25.5	25.6	25.3	25.7	25.4
	C	25.2	25.6	25.7	25.6	25.8	25.5
	D	25.1	25.5	25.7	25.7	25.7	25.2
Solvent control	A	24.9	25.1	25.5	25.1	25.4	25.2
	B	25.0	25.2	25.4	25.4	25.5	25.2
	C	25.0	25.0	25.4	25.4	25.8	25.6
	D	25.2	25.1	25.5	25.5	25.8	25.7
0.01	A	24.9	25.36	25.4	25.3	25.6	25.6
	B	24.8	25.3	25.4	25.3	25.6	25.7
	C	25.2	25.2	25.5	25.5	25.4	25.7
	D	25.2	25.1	25.5	25.5	25.7	25.9
0.03	A	24.7	25.3	25.5	25.3	25.3	25.7
	B	24.9	25.3	25.6	25.5	25.6	25.7
	C	25.2	25.2	25.6	25.4	25.6	25.6
	D	25.1	25.3	25.6	25.5	25.4	25.7
0.1	A	24.6	25.5	25.5	25.3	25.6	25.7
	B	24.7	25.5	25.5	25.5	25.6	25.6
	C	25.0	25.3	25.6	25.5	25.6	25.8
	D	25.2	25.6	25.6	25.6	25.6	25.8
0.32	A	24.9	25.3	25.4	25.2	25.4	25.6
	B	24.9	25.2	25.5	25.4	25.5	25.8
	C	25.0	25.1	25.5	25.4	25.5	25.6
	D	25.1	25.3	25.5	25.4	25.5	25.6
1.0	A	24.8	25.5	25.3	25.3	25.4	25.6
	B	24.8	25.4	25.5	25.4	25.4	25.5
	C	25.2	25.2	25.5	25.2	25.5	25.7
	D	25.1	25.4	25.6	25.4	25.6	25.7

All measurements are quoted to 1 decimal place

 

Continuous temperature monitoring summary

Time period (Days)	Continuous temperature data (°C)
	Mean Current Temperature	Minimum Temperature	Maximum Temperature
0 – 5 (hatch day)	25.4	25.0	25.6
6 – 12	25.6	25.5	25.8
13 – 19	25.7	25.5	25.8
20 – 26	25.8	25.4	25.9
27 – 33	25.6	25.3	25.8
Pooled	25.6	25.0	25.9

Max/Min thermometer checked against a calibrated liquid-in-glass thermometer (±0.2°C).

Continuous monitoring data recorded from DWC Replicate D.

All measurements are quoted to 1 decimal place.

Validity criteria fulfilled:

yes

Conclusions:

The overall NOEC, based on nominal concentrations of 6-tert-butyl-2,4-xylenol at Day 28 post-hatch was 0.32 mg/L.

Executive summary:

Test substance: 6-tert-butyl-2,4-xylenol

Subject: 6-tert-butyl-2,4-xylenol: Determination of effects on the Early Life-Stage of the fathead minnow (Pimephales promelas)

Guideline: OECD Test Guideline (TG; 2013), Test No. 210: Fish, Early-life Stage Toxicity Test

Test species: Newly fertilised (<24 hours old) eggs of Pimephales promelas

Source of organisms: Eggs supplied from broodstock held at Scymaris Ltd

Test concentrations: Dilution water control, solvent control and nominal concentrations of 0.01, 0.03, 0.1, 0.32 and 1.0 mg/L

Test design: Flow-through dosing design with 4 replicates per treatment

Exposure duration: egg to 28 days post-hatch

Nominal test temperature: 25 ± 1.5°C

Results based on nominal concentrations of 6-tert-butyl-2,4-xylenol

Endpoint: Hatch

	6-tert-butyl-2,4-xylenol (mg/L)	95% confidence limits
NOEC	≥1.0	N/A
LOEC	>1.0	N/A
EC10	N.D. (>1.0)	N/A
EC20	N.D. (>1.0)	N/A
EC50	N.D. (>1.0)	N/A

N.D. = Not determined

 

Endpoint: Survival (post-hatch)

	6-tert-butyl-2,4-xylenol (mg/L)	95% confidence limits
NOEC	≥1.0	N/A
LOEC	>1.0	N/A
EC10	N.D. (>1.0)	N/A
EC20	N.D. (>1.0)	N/A
EC50	N.D. (>1.0)	N/A

N.D. = Not determined

 

Endpoint: Growth (length)

	6-tert-butyl-2,4-xylenol (mg/L)	95% confidence limits
NOEC	0.32	N/A
LOEC	1.0	N/A
EC10	>1.0	N/A
EC20	>1.0	N/A
EC50	>1.0	N/A

 

Endpoint: Growth (wet weight)

	6-tert-butyl-2,4-xylenol (mg/L)	95% confidence limits
NOEC	0.32	N/A
LOEC	1.0	N/A
EC10	0.493	0.187-1.30
EC20	0.931	0.306-2.87
EC50	>1.0	N/A

 

Endpoint: Growth (dry weight)

	6-tert-butyl-2,4-xylenol (mg/L)	95% confidence limits
NOEC	0.32	N/A
LOEC	1.0	N/A
EC10	0.507	0.206-1.25
EC20	0.977	0.326-2.83
EC50	>1.0	N/A

 

The overall NOEC, based on nominal concentrations of 6-tert-butyl-2,4-xylenol at Day 28 post-hatch was therefore 0.32 mg/L.

Description of key information

OECD 210 - Long term toxicity to Fish

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.32 mg/L