4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

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:

17 June to 20 July 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

yes

Remarks:

See "Any other information" or details.

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:

Samples were collected by taking 10 mL with glass pipettes into disposable glass vials containing a 10 mL of acetonitrile.
Samples with a nominal concentration ≤4 μg/L were diluted 1:1 with acetonitrile, as described above.
Samples with a nominal concentration >4 μg/L were diluted 1:1 with acetonitrile as described above, and further diluted with 50:50 acetonitrile/water to within the calibration range.

Vehicle:

yes

Remarks:

Tri Ethylene Glycol (TEG)

Details on test solutions:

This study was run with a dilution water control (DWC), solvent control (SC) and nominal 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline concentrations of 30, 40 and 50 μg/L.
Dosing stocks were prepared by dissolving a known amount of 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in 50 mL of TEG in a volumetric flask. A stock was prepared of only TEG in a volumetric flask for the SC. All stocks were treated the same, with shaking and 30 minute ultrasonication used to dissolved the 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline 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 DWC contained dilution water only with no solvent addition. The SC and the and nominal concentrations 30, 40 and 50 μg/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 exposure Days 0, 5 (hatch Day),12, 19, 26, and 33.

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 daily. 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, sieved prior to addition to remove seawater) were fed from post-hatch Day 2. A pelleted commercial food, fed ad libitum from Day 16 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.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

28 d

Remarks on exposure duration:

Exposure duration: egg to 28 days post-hatch

Post exposure observation period:

No post exposure observation period sepcified

Hardness:

Measurements of the water hardness were conducted on the DWC, SC and nominal 50 μg/L treatment at test start and end. The water hardness measured test solutions at test start and end ranged from 56.33 to 62.66 mg/L CaCO3.

Test temperature:

The temperature of the test solutions measured on six occasions throughout the study ranged from 24.0 to 24.7°C.

pH:

The pH values of the test solutions, measured throughout the study ranged from 7.28 to 7.76.

Dissolved oxygen:

The dissolved oxygen (DO) concentrations of the test solutions, measured on thirteen occasions throughout the study ranged from 86.4 to 99.5% of the air saturation value (ASV)

Salinity:

Not applicable

Conductivity:

228 μS/cm

Nominal and measured concentrations:

Dilution water control (DWC), Solvent control (SC) and nominal concentrations 30, 40 and 50 μg/L

Details on test conditions:

Test procedure
The dosing of 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline was started on the 14 June 2021. The exposure commenced on 17 June 2021 by placing a nominal 20 embryos, <24 hours old, into the incubation cup in each replicate test tank (nominal 80 embryos in each of the DWC, SC and nominal concentrations 30, 40 and 50 μg/L). Embryos from 5 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 all 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.

Feeding regime
The hatched larvae/fry were fed daily. 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, sieved prior to addition to remove seawater) were fed from post-hatch Day 2. A pelleted commercial food, fed ad libitum from Day 16 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 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.

Analytical Method
To confirm the actual exposure concentrations being achieved, the concentration of 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in the test solutions were measured using the LC-MS/MS method detailed

Dilution water
The dilution water was town’s water, which had been coarsely filtered to remove particulate material, passed through activated carbon and dechlorinated using a sodium thiosulphate solution. Salts were added, as required, to maintain minimum hardness levels, and the treated water passed through an ultraviolet steriliser and filtered to 10 μm. The supply was then delivered to a temperature-controlled tank adjacent to the test laboratory which was set to the nominal test temperature of 25 ± 1.5°C. It was finally re-filtered to ≤5 μm, in the laboratory, before distribution to the test rigs.
The dechlorinated water is regularly monitored for routine parameters (e.g. pH, hardness, conductivity, alkalinity). In addition, the dechlorinated supply is periodically monitored for ammonia, total organic carbon, chemical oxygen demand and suspended solids plus a range of trace metals, pesticides and PCBs.

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. Where possible, any materials, other than glass, that were in contact with test solution were restricted to inert plastics or silicone.
A flow-through test system was used for this study. There was a ~5.5 hour period on exposure Day 6 where the dosing and water flow was stopped and the tanks were kept as a static system. This was because the heating system required emergency repairs. Due to the relatively short period that the flow-through system was stopped (in context to the total study duration) and as the temperature remained within 25 ± 1.5°C throughout the affected period, this was not thought to have affected the outcome of the test.
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 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 a mixing/flow splitting chambers at a nominal rate of 400 mL/min. All chambers, with the exception of the controls, also received the required amount of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline 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 0, 12, 19, and 26 over a known time interval. Measurements of the dilution water delivery and aquaria inflow rates were made on Day 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. The dilution water flow rates were maintained within ±10% of nominal. The aquaria flow rates were checked weekly and maintained within ±20% through out the study, both within each aquarium and between the aquaria for the duration of the test.
The fish stocking density did not exceed 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.

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 NOEC, the concentration of 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline observed to have no significant effect (p<0.05), was determined directly from the test observations as well as statistical analysis to determine if it was 0 biologically meaningful.

Hatching and Survival post-hatch
Replicate data were pooled and the DWC and SC data for hatch and survival post-hatch were compared by Fisher’s Exact Binomial Test. The DWC and SC were then pooled and compared to the 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline treatment concentrations for hatch and survival post-hatch data using Chi2 2x2 Table Test with Bonferroni Correction.

Length and weight
Comparisons of total length, wet weight and dry weight were made between the DWC and the solvent control, and the pooled controls and the 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline treatment concentrations. All sets of individual fry data (replicates pooled) were assessed for normality and equality of variance.

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 0, 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 DWC, SC and nominal concentration 50 μg/L on exposure Day 0 and on Day 28 post-hatch.
The light intensity at aquaria water level in approximately the middle of the test rig was measured on a single occasion during the test.

Reference substance (positive control):

not required

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 50 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 50 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 50 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Details on results:

Analytical data
All analytical values are quoted to three significant figures and percentages to the nearest integer.
A single replicate test vessel from each treatment was sampled and measured at each time point. The limit of quantification of 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline was 0.125 μg/L taken from the method validation study.
The DWC and SC groups were measured and determined to be Measured concentrations of the fortified recovery samples were 68-134% of nominal. The mean measured concentration of the fortified recovery samples were 78-117% demonstrating accuracy of the method.

Biological data
Behaviour and Appearance
Throughout the study, no unusual of observations of fry behaviour or appearance were recorded. One fry with a bent spine exhibiting a lack of development, but otherwise healthy, was noted to be present in replicate A of the nominal 50 μg/L treatment on Day 28 post-hatch.

Control comparison
No significant differences (p < 0.05) were found between the DWC and the SC groups for any of the endpoints (hatch, survival, length, wet weight or dry weight), so the controls were pooled and all statistical comparisons were carried out between the pooled controls and 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline treatment groups.

Hatch data
Hatch Day in this study was 22 June 2021, which was exposure Day 5. Due to an error in counting at the beginning of the study, the number of fish hatched at the end of the study was calculated from the number of fish surviving at the end of the study, with the fish that died during the post-hatch period taken away. This prevented the results from being skewed due to the miss count of the fish during the study and did not affect the outcome of the study.
The range of percentage hatch success across all replicates was 85-100%. The mean percentage hatch success across all replicates in the DWC and SC were 94 and 93%, respectively. No significant difference (p < 0.05) was found between the DWC and SC groups (Chi2 2x2 table). No significant differences (p < 0.05) were found between the pooled controls and the treatment concentrations for hatch data (Chi2 Table Test after Bonferroni correction). The LOEC for hatch was therefore >50 μg/L, and the NOEC was ≥50 μg/L.

Survival data
The range of percentage survival post-hatch across all replicates was 94-100%. The mean percentage survival post-hatch in the DWC and SC were 100 and 95%, respectively. No significant differences (p > 0.05) were found between the pooled controls and the treatment concentrations (Chi2 Table Test after Bonferroni correction). The LOEC for survival was therefore >50 μg/L, and the NOEC was ≥50 μg/L.

Length data
The total lengths of the fry in the DWC ranged from 18.47 to 28.21 mm with a mean of 24.98 mm. The total lengths of the fry in the SC ranged from 21.83 to 29.16 mm with a mean of 25.27 mm. The mean length of the DWC and SC both exceeded the 18 mm typical control minimum mean total length requirement of the OECD 210 TG (Ref 1). No significant difference (at p <0.05) was found between the DWC and the SC groups (Student-t test for homogenous variances). No significant differences (p < 0.05) were found between the length of the pooled controls and any of the test concentrations (Dunnett’s Multiple t-test). The LOEC for total length was therefore >50 μg/L, and the NOEC was ≥50 μg/L.

Weight data
The wet weights of the fry in the DWC ranged from 0.05477 to 0.20910 g with a mean of 0.13880 g. The wet weights of the fry in the SC ranged from 0.07572 to 0.22908 g with a mean of 0.14744 g. No significant difference (at p <0.05) was found between the DWC and the SC groups (Student-t test for homogenous variances). No significant differences were found between the wet weight in the pooled controls and any of the test concentrations (Dunnett’s Multiple t-test procedure). The LOEC for wet weight was therefore >50 μg/L, and the NOEC was ≥50 μg/L.
The summary dry weight data are shown in Table 6. The dry weights of pooled fry in the DWC ranged from 0.02963 to 0.03380 g with a mean of 0.03139 g. The dry weights of pooled fry in the SC ranged from 0.02768 to 0.03724 g with a mean of 0.03188 g. No significant difference (at p <0.05) was found between the DWC and the SC groups (Student-t test for homogenous variances).
During the calculation of the dry weights, pooled replicate D of the nominal 40 μg/L concentration resulted in a negative number and replicate C of the nominal 40 μg/L concentration had a value double what was seen in replicates A and B, despite similar numbers of fish. It is thought that during the weighing of the vials prior to the fish being added, 40 μg/L C and D were inadvertently switched around. However, due to not being able to be definitively determine the cause, it was decided to remove these two replicates from the calculation of the dry weights. This decision did not affect the outcome of the test as it ensured the data was not skewed by this notation error.
No significant differences (p < 0.05) were found between the dry weight of the pooled controls and any of the test concentrations (Dunnett’s Multiple t-test procedure). The LOEC for dry weight was therefore >50 μg/L, and the NOEC was ≥50 μg/L.

Physical and chemical data
The pH values of the test solutions, measured throughout the study ranged from 7.28 to 7.76.
The dissolved oxygen (DO) concentrations of the test solutions, measured on thirteen occasions throughout the study ranged from 86.4 to 99.5% of the air saturation value (ASV).
Measurements of the water hardness were conducted on the DWC, SC and nominal 50 μg/L treatment at test start and end. The water hardness measured test solutions at test start and end ranged from 56.33 to 62.66 mg/L CaCO3.
The temperature of the test solutions measured on six occasions throughout the study ranged from 24.0 to 24.7°C.
The continuous temperature measurement, recorded in the DWC Replicate D ranged from 23.6 – 25.3°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 25 lux (cosine).

Aquaria flow rates
The overall range of the aquaria flow rates, measured for each test aquaria throughout the exposure ranged from 96 to 104 mL/min and had an overall mean of 99 mL/min. This mean value provides a nominal 15.05 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 and the volume recorded for each aquaria. The stocking density in each aquaria at test end was below the maximum permitted 5 g/L wet weight, with a range of 0.016 – 0.021 g/L wet weight.

Results with reference substance (positive control):

Not required

Reported statistics and error estimates:

All biological analysis was performed using the statistical package ToxRat

 Analytical results

	Measured Concentrations of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in Dechlorinated Water
Nominal conc. (µg/L)	Day -2		Day 0		Hatch Day 0		Post-Hatch Day 7		Post-Hatch Day 14		Post-Hatch Day 21		Post-Hatch Day 28
	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal
DWC	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A
SC	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A	<LOQ	N/A
30	27.3	91	25.4	85	33.0	110	25.1	84	30.3	101	24.7	82	25.6	85
40	35.2	88	39.1	98	38.7	97	36.2	91	36.3	91	39.5	99	37.8	95
50	45.4	91	51.1	102	48.0	96	44.7	89	49.6	99	49.0	98	48.8	98

All measurements are quoted to 3 significant figures. Percentages to nearest integers.

The limit of quantification (LOQ) was 0.125 µg/L.

 

Fortified Solutions

	Measured Concentrations of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in Dechlorinated Water
Nominal conc. (µg/L)	Day -2		Day 0		Hatch Day 0		Post-Hatch Day 7		Post-Hatch Day 14		Post-Hatch Day 21		Post-Hatch Day 28
	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal	(µg/L)	% of nominal
0.500	0.459	92	0.542	108	0.537	107	0.472	94	0.451	90	0.528	106	0.570	114
0.500	0.364	73	0.394	79	0.539	108	0.502	100	0.446	89	0.426	85	0.672	134*
0.500	0.342	68*	0.483	97	0.568	114	0.497	99	0.416	83	0.390	78	0.515	103
Mean	0.388	78	0.473	95	0.548	110	0.490	98	0.438	88	0.448	90	0.586	117
SD	0.062	12	0.074	15	0.017	3	0.016	3	0.019	4	0.071	14	0.080	16

All measurements are quoted to 3 significant figures. Percentages to nearest integers.

The limit of quantification (LOQ) was 0.125 µg/L.

*Due to suspected error in preparation, samples failed to meet the acceptance criteria. Samples were not outliners therefore are included in the mean calculation.

 

Hatch and survival data

Nominal conc. of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Replicate	Eggs introduced Day 0	Total number hatched	% Hatched		Number of fry surviving	% Survival post-hatch
				Replicate	Treatment		Replicate	Treatment
DWC	A	20	17	85	94	17	100	100
	B	20	19	95		19	100
	C	20	19	95		19	100
	D	20	20	100		20	100
SC	A	20	16	80	93	15	94	95
	B	20	19	95		18	95
	C	20	19	95		17	89
	D	20	20	100		20	100
30	A	23	21	91	93	21	100	99
	B	20	18	90		17	94
	C	22	21	95		21	100
	D	20	19	95		19	100
40	A	22	20	91	94	19	95	95
	B	20	18	90		18	100
	C	20	19	95		18	95
	D	20	20	100		18	90
50	A	20	20	100	96	19	95	97
	B	20	20	100		19	95
	C	20	18	90		18	100
	D	20	19	95		19	100

No statistically significant (p >0.05) differences between the pooled controls (DWC and SC) and the treatment groups were observed for Hatch or Survival post-hatch data. Percentages are quoted to the nearest integer.

 

Summary length data

Nominal conc. of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Replicate	Number	Mean	Minimum	Maximum	Standard deviation
DWC	A	17	25.17	18.47	28.21	2.31
	B	19	24.48	22.88	26.13	1.10
	C	19	25.12	22.16	26.98	1.24
	D	20	25.16	23.51	26.93	1.15
	Pooled	75	24.98	18.47	28.21	1.50
SC	A	15	25.54	22.27	27.58	1.52
	B	18	24.58	21.83	26.63	1.54
	C	17	26.51	24.32	29.16	1.43
	D	20	24.63	22.36	27.57	1.49
	Pooled	70	25.27	21.83	29.16	1.67
30	A	21	24.89	22.72	26.88	1.16
	B	17	25.77	23.02	28.11	1.51
	C	21	24.46	20.28	28.19	1.94
	D	19	25.38	22.36	28.06	1.40
	Pooled	78	25.08	20.28	28.19	1.58
40	A	19	25.60	21.77	27.41	1.39
	B	18	25.12	19.71	26.74	1.92
	C	18	25.99	23.71	28.09	1.30
	D	18	25.28	21.82	27.30	1.55
	Pooled	73	25.50	19.71	28.09	1.56
50	A	19	24.90	11.19	27.11	3.50
	B	19	25.07	22.87	27.52	1.29
	C	18	25.31	22.27	27.63	1.34
	D	19	24.50	22.82	26.43	1.08
	Pooled	75	24.94	11.19	27.63	2.04

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

 

Summary wet weight data

Nominal conc. of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Replicate	Number	Mean	Minimum	Maximum	Standard deviation
DWC	A	17	0.14534	0.05477	0.20650	0.03630
	B	19	0.13110	0.08778	0.16840	0.02184
	C	19	0.13551	0.09197	0.20910	0.02641
	D	20	0.14366	0.11124	0.18047	0.02153
	Pooled	75	0.13880	0.05477	0.20910	0.02690
SC	A	15	0.15994	0.10181	0.20170	0.02797
	B	18	0.13018	0.07572	0.18078	0.03235
	C	17	0.17314	0.12040	0.22908	0.03241
	D	20	0.13175	0.08595	0.17602	0.02479
	Pooled	70	0.14744	0.07572	0.22908	0.03428
30	A	21	0.13900	0.10104	0.18736	0.02410
	B	17	0.15768	0.11573	0.22022	0.02998
	C	21	0.13402	0.06551	0.22799	0.03479
	D	19	0.14382	0.10019	0.20917	0.02997
	Pooled	78	0.14371	0.06551	0.22799	0.03059
40	A	19	0.14608	0.08750	0.18189	0.02232
	B	18	0.15073	0.06879	0.17979	0.02690
	C	18	0.15496	0.10476	0.22045	0.02868
	D	18	0.14061	0.09100	0.18235	0.02462
	Pooled	73	0.14743	0.06879	0.22045	0.02567
50	A	19	0.14721	0.00719	0.18270	0.03954
	B	19	0.13987	0.09811	0.20026	0.02390
	C	18	0.15395	0.11795	0.20722	0.02638
	D	19	0.13049	0.09967	0.17090	0.01967
	Pooled	75	0.14273	0.00719	0.20722	0.02915

No statistically significant (p <0.05) differences between the pooled controls and the treatment groups were observed for wet weight data.

 

Dry weight data

Nominal conc. of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Replicate	Mean dry weight per fish	Total treatment dry weight mean	Standard deviation
DWC	A	0.03380	0.03139	0.00100
	B	0.02963
	C	0.03116
	D	0.03098
SC	A	0.03446	0.03188	0.00472
	B	0.02816
	C	0.03724
	D	0.02768
30	A	0.02841	0.02999	0.00233
	B	0.03262
	C	0.02769
	D	0.03124
40	A	0.03138	0.03158	0.00028
	B	0.03178
	C	-*
	D	-*
50	A	0.03163	0.03040	0.00224
	B	0.02971
	C	0.03269
	D	0.02760

No statistically significant (p <0.05) differences between the pooled controls and the treatment groups were observed for dry weight data.

*Nominal treatment 40 µg/L replicates C and D excluded from analysis.

 

Test vessel pH values

Nominal conc. of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Replicate	pH
		Day 0	Hatch Day	Day 7 post-hatch	Day 14 post-hatch	Day 21 post-hatch	Day 28 post-hatch
DWC	A	7.49	7.37	7.40	7.51	7.33	7.38
	B	7.57	7.40	7.41	7.57	7.35	7.42
	C	7.64	7.46	7.43	7.54	7.37	7.42
	D	7.69	7.47	7.49	7.52	7.40	7.49
SC	A	7.70	7.53	7.44	7.55	7.34	7.44
	B	7.72	7.56	7.48	7.58	7.38	7.47
	C	7.61	7.52	7.42	7.53	7.33	7.44
	D	7.67	7.53	7.52	7.55	7.38	7.47
30	A	7.67	7.53	7.43	7.55	7.35	7.41
	B	7.68	7.56	7.46	7.55	7.34	7.41
	C	7.70	7.53	7.46	7.55	7.36	7.47
	D	7.69	7.55	7.43	7.56	7.39	7.47
40	A	7.67	7.52	7.41	7.53	7.28	7.44
	B	7.72	7.53	7.45	7.54	7.32	7.45
	C	7.69	7.57	7.44	7.53	7.30	7.46
	D	7.76	7.60	7.48	7.57	7.35	7.48
50	A	7.71	7.58	7.51	7.56	7.35	7.48
	B	7.69	7.62	7.50	7.52	7.30	7.45
	C	7.76	7.58	7.44	7.54	7.33	7.45
	D	7.63	7.54	7.46	7.53	7.34	7.45

All measurements are quoted to 3 significant figures.

 

Test vessel dissolved oxygen (DO) concentrations

Nominal conc. of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/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
DWC	A	88.9	94.4	96.1	94.9	88.9	92.1
	B	89.6	95.1	95.0	95.9	89.0	92.0
	C	89.0	95.2	94.7	96.7	91.3	91.0
	D	88.3	92.5	95.4	94.1	89.1	88.2
SC	A	88.6	94.6	94.2	95.6	89.6	89.0
	B	89.6	94.1	95.0	96.2	89.1	88.4
	C	88.36	95.0	95.8	94.9	89.2	88.4
	D	88.6	94.1	99.5	94.7	90.1	89.4
30	A	88.1	94.5	94.3	94.7	88.5	89.0
	B	88.4	94.2	94.4	93.9	87.9	87.1
	C	88.1	94.4	94.9	95.1	90.4	88.0
	D	88.9	93.9	94.4	95.6	90.5	88.1
40	A	88.9	93.5	93.7	94.2	88.4	88.2
	B	88.5	94.0	94.9	95.4	89.6	87.4
	C	87.8	94.1	93.9	94.2	90.2	86.7
	D	88.0	93.6	92.1	94.1	90.4	88.4
50	A	88.2	93.5	92.2	93.8	89.1	87.4
	B	88.0	93.6	91.9	93.0	88.4	87.1
	C	87.2	93.7	93.7	92.9	89.3	86.4
	D	87.3	93.5	92.9	93.0	88.5	86.8

All measurements are quoted to 1 decimal place.

 

Test vessel pH values

Nominal conc. of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (µg/L)	Replicate	Temperature (°C)
		Day 0	Hatch Day	Day 7 post-hatch	Day 14 post-hatch	Day 21 post-hatch	Day 28 post-hatch
DWC	A	24.7	24.3	24.4	24.4	24.3	24.1
	B	24.7	24.2	24.4	24.4	24.2	24.3
	C	24.7	24.3	24.4	24.4	24.3	24.2
	D	24.6	24.3	24.4	24.3	24.4	24.1
SC	A	24.7	24.4	24.5	24.3	24.4	24.2
	B	24.7	24.4	24.5	24.36	24.4	24.2
	C	24.7	24.4	24.5	24.3	24.3	24.1
	D	24.6	24.4	24.5	24.3	24.3	24.2
30	A	24.6	24.3	24.5	24.3	24.5	24.1
	B	24.5	24.3	24.6	24.3	24.5	24.2
	C	24.5	24.2	24.4	24.3	24.4	24.1
	D	24.5	24.3	24.3	24.3	24.4	24.1
40	A	24.6	24.4	24.5	24.3	24.3	24.1
	B	24.7	24.4	24.4	24.4	24.4	24.2
	C	24.7	24.2	24.5	24.4	24.3	24.2
	D	24.7	24.3	24.4	24.3	24.3	24.1
50	A	24.5	24.3	24.4	24.3	24.2	24.1
	B	24.5	24.2	24.6	24.4	24.4	24.1
	C	24.5	24.2	24.6	24.3	24.4	24.1
	D	24.5	24.3	24.5	24.4	24.3	24.0

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 – hatch	25.2	25.0	25.3
1 – 7 post-hatch	24.8	23.6	25.2
8 – 14 post-hatch	24.9	24.8	25.0
15 – 21 post-hatch	24.9	24.8	25.1
22 – 28 post-hatch	24.9	24.8	25.1
Pooled	24.9	23.6	25.3

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

Continuous monitoring data are recorded from DWC Replicate D

All measurements are quoted to 1 decimal place

Validity criteria fulfilled:

yes

Conclusions:

Including all of the assessed endpoints at the end of the study, and using the nominal concentrations, the overall LOEC for 4-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in this study was >50 μg/L, and the overall NOEC was ≥50 μg/L.

Executive summary:

Subject: 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline: 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

Test concentrations: Dilution water control (DWC), Solvent control (SC) and nominal concentrations 30, 40 and 50 μg/L

Test design: Extended limit test, Flow-through dosing design, 4 replicates per treatment,

Exposure duration: egg to 28 days post-hatch

Nominal test temperature: 25 ± 1.5°C

 

Results based on nominal conc. of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Endpoint: Hatch: NOEC: ≥50 μg/L

Endpoint: Survival (post-hatch) NOEC: ≥50 μg/L

Endpoint: Growth (length) NOEC: ≥50 μg/L

Endpoint: Growth (wet weight) NOEC: ≥50 μg/L

Endpoint: Growth (dry weight) NOEC: ≥50 μg/L

 

The overall NOEC, based on nominal concentrations of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline at Day 28 post-hatch was therefore ≥50 μg/L.  No toxicity was observed at the limit of solubility in water.

Description of key information

The overall NOEC, based on nominal concentrations of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline at Day 28 post-hatch was therefore ≥50 μg/L.  There was no toxicity at the limit of solubility in water.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

50 µg/L

Additional information

Subject: 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline: 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

Test concentrations: Dilution water control (DWC), Solvent control (SC) and nominal concentrations 30, 40 and 50 μg/L

Test design: Extended limit test, Flow-through dosing design, 4 replicates per treatment,

Exposure duration: egg to 28 days post-hatch

Nominal test temperature: 25 ± 1.5°C

 

Results based on nominal conc. of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Endpoint: Hatch: NOEC: ≥50 μg/L

Endpoint: Survival (post-hatch) NOEC: ≥50 μg/L

Endpoint: Growth (length) NOEC: ≥50 μg/L

Endpoint: Growth (wet weight) NOEC: ≥50 μg/L

Endpoint: Growth (dry weight) NOEC: ≥50 μg/L

 

The overall NOEC, based on nominal concentrations of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline at Day 28 post-hatch was therefore ≥50 μg/L.  No toxicity was observed at the limit of solubility in water.