2,5-di-tert-pentylhydroquinone

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:

20 October 2017 to 10 July 2018.

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 Guideline for Testing of Chemicals No. 210 Fish, Early Life Stage Toxicity Test (July 2013)

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

Remarks:

Details of analytical information is appended below for reference.

Details on sampling:

Concentrations of LOWINOX® AH-25 were determined by diluting treated mains water samples with acetonitrile, then diluting further with acetonitrile/treated mains water, 1:4, v:v as required to bring the response within the calibration range. Samples are analysed by injection onto a liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) system.
The analyte is highly unstable, being extremely sensitive to oxidation. The method used measured both the parent analyte, LOWINOX® AH-25, and its oxidation product. LOWINOX® AH-25 was quantified against matrix-matched standards. True quantification of the oxidation product was not possible since no primary standard was available. The peak area ratio of the parent to the oxidation product was reported during sample analysis to provide an indication of the relative amounts present.
Samples (10 mL) from the control, solvent control and each test concentration were taken for analysis prior to exposure and on Days 0 and 3 (pre-hatch) and Days 0, 3, 7, 10, 13, 16, 20, 23 (samples discarded as could not be analysed due to issues with the analytical instrument), 24 and 28 (post-hatch). Analysis was conducted on one replicate per concentration with replicates sampled alternatively. At each sampling occasion, duplicate samples were taken. One for chemical analysis and one as a ‘back up’ sample should further analysis be required.

Vehicle:

yes

Details on test solutions:

A solvent stock solution of 2 mg/mL was prepared by dissolving ca 100 mg of test substance in 50 mL of acetone. Serial dilutions were prepared in acetone to give further solvent stock solutions of 0.64, 0.20, 0.064 and 0.020 mg/mL. The 0.020, 0.064, 0.20, 0.64 and 2 mg/mL solvent stock solutions were each separately dosed at a rate of 0.060 mL/hour to a diluent flow rate of 20 ± 10% mL/min to give the 1.0, 3.2, 10, 32 and 100 µg/L test concentrations, respectively.

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.

The eggs were deposited approximately 24 hours prior to testing and were collected in glass scintillation vials containing treated mains water. The embryos were acclimated to laboratory conditions for at least one hour prior to being added to the vessels. During this time the stage of embryonic development was assessed under a microscope.

Test type:

flow-through

Water media type:

freshwater

Remarks:

was treated mains water (treated via activated carbon particulate filter)

Limit test:

no

Total exposure duration:

28 d

Remarks on exposure duration:

As per test guideline.

Post exposure observation period:

Not required for the study

Hardness:

Not specified

Test temperature:

24.2 - 25.3 ºC

pH:

7.10 - 7.85

Dissolved oxygen:

90 - 105 % ASV

Salinity:

Not applicable - freshwater study

Conductivity:

200 uS/cm

Nominal and measured concentrations:

The geometric mean measured concentrations of parent test substance were calculated to be 2.1, 13, 31 and 44 µg/L for the 3.2, 10, 32 and 100 µg/L test concentrations, respectively

Details on test conditions:

Definitive Test
Information supplied by the Sponsor gave an acute LC50 value for fathead minnow of 0.12 mg/L. Based on this information, the definitive test was conducted at nominal concentrations of 1.0, 3.2, 10, 32 and 100 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 2 mg/mL was prepared by dissolving ca 100 mg of test substance in 50 mL of acetone. Serial dilutions were prepared in acetone to give further solvent stock solutions of 0.64, 0.20, 0.064 and 0.020 mg/mL.
The 0.020, 0.064, 0.20, 0.64 and 2 mg/mL solvent stock solutions were each separately dosed at a rate of 0.060 mL/hour to a diluent flow rate of 20 ± 10% mL/min to give the 1.0, 3.2, 10, 32 and 100 µg/L test concentrations, respectively.
A solvent control was prepared in a similar manner by dosing solvent only at a rate of 0.060 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.

Appearance of Test Media
The appearance, colour and behaviour of the test substance in the test media were recorded daily.

Test Procedures
The test tanks were nominal 3.5 L constructed glass aquariums. Four replicate tanks were used for the control, solvent control and each test concentration each filled with ca 3 L of the appropriate media.
Egg incubation chambers used in the test were 17 cm long glass cylinders (6.5 cm internal diameter) with a 0.5 - 1.0 mm non-reactive mesh attached to one end (bottom).
The glass cylinders had opposing holes approximately 6.5 cm from the top (at the opposite end to the mesh), through which a stainless steel rod was inserted to enable each chamber to be suspended across the width of each replicate test vessel. Each replicate test tank contained one incubation chamber.
To ensure adequate movement of water within the test vessels and maintain dissolved oxygen concentrations in the egg incubation chambers, two aeration tubes were added to each replicate vessel. The first was maintained in the test medium surrounding the incubation chamber and the second aerator was maintained within the incubation chamber, positioned with the tube end approximately 1 cm from the mesh on the end of the cylinder. The agitation/turbulence caused by the aerators on the water column within the chambers ensured that a dynamic equilibrium of concentration was achieved between the environment within the chamber and surrounding test medium. The turbulence created slight movement of the embryos and maintained good levels of aeration within the chamber.

Egg Addition and Observations
At the start of the test, groups of 20 eggs were added to glass scintillation vials half filled with treated mains water and each vial was randomly assigned to a test vessel.
The eggs were transferred using a discrete pipette into the corresponding incubation chamber. The eggs were released under the surface of the test media, as close to the mesh as possible.
During the pre-hatch period, where possible, non viable or necrotic eggs were removed, avoiding disturbance of adjacent viable eggs.
The post-hatch phase started once all of the viable eggs were considered to have hatched in the control groups. Once all viable eggs had hatched across all treatments and fish were actively feeding and mobile, the chambers were removed.
On Day 28 the total numbers of surviving larvae were counted and individual total fish lengths and wet weights of all remaining fish were determined. Prior to initial weighing, the fish were blotted dry to make sure that all surface water was removed.
The percentage post-hatch survival was determined by expressing the number of surviving larvae on Day 28 post-hatch as a percentage of the hatched larvae at Day 0 (post-hatch).

Feeding
The developing larvae were fed using brine shrimp (Artemia salinis) nauplii, freshly cultured during the test. Initially the larvae were fed approximately 24-hour old shrimp. From Day 6 post-hatch onwards the larvae were fed approximately 48-hour old shrimp.
Surplus feed and faecal material were siphoned from the test vessel on at least daily intervals during the test.

Water Quality and Environmental Conditions
The test was conducted in a light-controlled facility, with a 16-hour light:8 hour dark period with an approximate 30 minute dawn:dusk transition period.
The water temperature (°C), pH and dissolved oxygen concentration, expressed as a percentage of the air saturation value (% ASV), were measured at the start of the test and at weekly intervals in each test vessel.
Water temperatures (°C) were monitored continuously in a vessel maintained in the test area using a minimum/maximum digital thermometer.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

44 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Hatching succes, grwoth (length and weight)

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

2.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: post-hatch survival

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 44 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Hatching success, growth (length & weight)

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

13 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Post-hatch survival

Details on results:

Test Organism
The approximate age of the fertilised eggs on addition to the test vessels was ca 24 hours post-fertilisation. They were considered acceptable for use as their stage of embryonic development at the time of addition was at the early embryo/tail bud stage.

Definitive Test
Hatching Success
First egg hatch, in all treatment, solvent control and the control vessels occurred between Days 3 and 4 (post-egg addition). Completion of hatching occurred between Days 4 and 5 (post-egg addition) in all vessels. NOEC and LOEC values for time to first egg hatch and the egg-hatching period were therefore not statistically derived. This indicated no difference in the time to first hatch or hatching period across all treatments when compared to the control groups.
The mean hatching success of embryos in the control and solvent control vessels was 100% and 99% respectively, which was in excess of the validity criterion of ≥70%. It was therefore considered that the validation criterion had been achieved.
Mean hatching success in the treatments ranged between 98% - 100%. There were no statistically significant effects on hatching success at any of the test concentrations employed in the test compared to the combined controls. The NOEC and LOEC values for hatching success were considered to be 100 and >100 µg/L, respectively, based on nominal concentrations (44 and >44 µg/L, respectively, based on geometric mean measured concentrations of parent test substance).

Survival
The mean post-hatch survival in the control and solvent control groups was 88% and 76%, respectively, which was in excess of the validity criterion of ≥75%.
Mean post-hatch survival in the treatments ranged between 12% - 90%. Statistical analysis showed significant effects (p<0.05) in terms of post-hatch survival between the 10, 32 and 100 µg/L test concentrations and the combined controls. The NOEC and LOEC values for post-hatch survival were considered to be 3.2 and 10 µg/L, respectively, based on nominal concentrations (2.1 and 13 µg/L, respectively, based on geometric mean measured concentrations of parent test substance).

Fish Total Lengths and Wet Weights
No significant differences were observed between the combined controls and test groups in terms of length and wet weight.
Taking both length and weight into consideration, the NOEC and LOEC values for growth were considered to be 100 and >100 µg/L, respectively, based on nominal concentrations (44 and >44 µg/L, respectively, based on geometric mean measured concentrations of parent test substance).

Abnormalities
A small number of fish were observed to be small or pale or have a bent spine. However, these observations were not concentration specific and were therefore not considered to be due to any toxic effect but rather natural variation within the population.

Results with reference substance (positive control):

Not performed

Reported statistics and error estimates:

No statistically significant differences were observed in terms of hatching success or final length and wet weight at any of the test concentrations employed in the test compared to the combined controls. Statistically significant differences were observed in terms of post-hatch survival at the 10, 32 and 100 µg/L test concentrations compared to the combined controls.

Hatching Success

The results for hatching success are presented in Table 4 and summarised below.

Nominal concentration (µg/L)	Geometric mean measured concentration (µg/L)	Hatching success (%)
Control	Control	100
Solvent control	Solvent control	99
1.0	-	100
3.2	2.1^a	100
10	13	100
32	31	98
100	44	98

- Not calculated
^aMeasured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution

 

Survival

The results for survival of hatched larvae at Day 28 post-hatch are presented in Table 4 and summarised below.

Nominal concentration (µg/L)	Geometric mean measured concentration (µg/L)	Post-hatch survival Day 28 (%)
Control	Control	88
Solvent control	Solvent control	76
1.0	-	90
3.2	2.1^a	73
10	13	58*
32	31	51*
100	44	12*

- Not calculated
^aMeasured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution

* Significant (p <0.05) difference compared to the solvent control

 

Measured concentrations of LOWINOX® AH-25 in test media during the definitive test

Nominal concentration (µg/L)	Measured concentration (µg/L)
	Day 0 (pre-hatch)*	Day 3 (pre-hatch)	Day 0 (post-hatch)	Day 3 (post-hatch)*	Day 7 (post-hatch)*	Day 10 (post-hatch)
Control	-	-	-	-	-	-
Solvent control	-	-	-	-	-	-
1.0	0.14	0	0.35	0.23	0	0
3.2	0.79	4.28	1.72	0	0.58	2.46
10	6.4	8.83	6.8	6.81	10.9	102
32	0	97.1	25.2	22	17.6	29.1
100	26.7	26.3	34.2	43.4	50.3	67.2

– Non detected above limit of quantification (LOQ), assessed as 5 µg/L
* Analytical batch failed acceptance criteria. Results have been provided for information only and were not used in the calculation of the geometric mean measured concentrations

 

Nominal concentration (µg/L)	Measured concentration µg/L)					Geometric mean measured concentration (µg/L)
	Day 13 (post-hatch)	Day 16 (post-hatch)*	Day 20 (post-hatch)	Day 24 (post-hatch)*	Day 28 (post-hatch)
Control	-	-	-	-	-	-
Solvent control	-	-	-	-	-	-
1.0	0.24	0	0	0.69	<0	NC
3.2	0	1.5	0.78	4.56	<0	2.1^a
10	9.3	10.7	10.7	10.8	6.76	13
32	37.9	29.3	15.2	34.3	22.8	31
100	25.6	34.1	88.3	77	52.8	44

– Non detected above limit of quantification (LOQ), assessed as 5 µg/L
* Analytical batch failed acceptance criteria. Results have been provided for information only and were not used in the calculation of the geometric mean measured concentrations

NC = Not calculated
^aMeasured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ where no test substance was detected. Result should be treated with caution

 

Values for the 1.0 and 3.2 µg/L have been provided for information purposes only as values were <LOQ.

 

Water quality determinations during the fish early life stage test

Nominal concentration (µg/L)	Day
	Day 0 pre-hatch	Day 3 post-hatch	Day 10 post-hatch	Day 17 post-hatch	Day 24 post-hatch	Day 28 post-hatch
Temperature (ºC)
Control	24.5 – 24.9	24.9 – 25.0	25.1 – 25.3	24.9 – 25.1	24.7 – 24.8	24.6 – 24.8
Solvent control	24.4 – 24.7	24.8 – 25.0	24.8 – 25.1	24.7 – 25.1	24.7 – 24.9	24.4 – 24.7
1.0	24.6 – 24.8	24.7 – 24.8	24.8 – 25.1	24.9 – 25.1	24.8 – 24.9	24.4 – 24.7
3.2	24.4 – 24.7	24.5 – 24.7	24.9 – 25.0	24.9 – 25.1	24.9 – 25.2	24.6
10	24.2 – 24.8	24.4 – 24.8	24.9 – 25.1	24.9 – 25.2	24.9 – 25.0	24.5 – 24.7
32	24.3 – 24.6	24.5 – 24.7	24.7 – 24.9	24.9 – 25.2	24.9 – 25.0	24.5 – 24.9
100	24.2 – 24.3	24.2 – 24.4	24.6 – 24.8	24.9 – 25.0	24.7 – 24.8	24.5 – 24.6
pH
Control	7.67 – 7.72	7.55 – 7.63	7.33 – 7.39	7.76 – 7.85	7.20 – 7.29	7.47 – 7.58
Solvent control	7.53 – 7.63	7.50 – 7.54	7.24 – 7.38	7.66 – 7.78	7.19 – 7.32	7.44 – 7.57
1.0	7.61 – 7.67	7.52 – 7.57	7.26 – 7.28	7.61 – 7.71	7.21 – 7.25	7.44 – 7.48
3.2	7.65 – 7.67	7.57 – 7.59	7.27 – 7.34	7.65 – 7.74	7.12 – 7.32	7.40 – 7.43
10	7.65 – 7.66	7.51 – 7.53	7.26 – 7.33	7.65 – 7.70	7.20 – 7.28	7.33 – 7.38
32	7.58 – 7.68	7.51 – 7.54	7.34 – 7.35	7.60 – 7.66	7.10 – 7.21	7.42 – 7.49
100	7.63 – 7.69	7.55 – 7.57	7.40 – 7.46	7.62 – 7.71	7.25 – 7.30	7.34 – 7.35
Dissolved oxygen concentration (%ASV)
Control	101 – 104	100	99	98 – 105	94 – 98	93 – 99
Solvent control	101 – 102	98 – 99	82 – 99	99 – 102	92 – 101	92 – 98
1.0	102 – 103	99 – 100	93 – 94	88 – 103	92 – 94	93 – 97
3.2	101 – 103	100 – 101	92 – 96	98 – 108	90 – 102	94 – 96
10	101 – 103	94 – 99	87 – 95	91 – 103	90 – 97	90 – 94
32	100 – 102	97 – 99	95 – 96	92 – 102	88 – 92	94 – 98
100	100 – 101	96 – 97	96 – 101	94 – 96	92 – 98	86 – 89

ASV – Air Saturation Value
Values stated are the range across the four replicates

 

Continuously recorded temperature range during the fish early life stage test

Day number	*Recorded Temperature (°C)
0	25.2
1	25.1 – 25.6
2	25.1 – 25.6
3	25.2 – 25.6
4	25.2 – 25.8
5	24.9 – 25.3
6	25.5 – 25.9
7	25.0 – 25.5
8	24.9 – 25.6
9	25.1 – 25.4
10	25.0 – 25.6
11	25.0 – 25.8
12	25.0 – 25.4
13	25.2 – 25.8
14	25.1 – 25.7
15	25.2 – 25.7
16	25.2 – 25.7
17	25.2 – 25.7
18	25.3 – 25.7
19	25.5 – 25.7
20	25.5 – 25.7
21	25.3 – 25.8
22	25.3 – 25.6
23	25.4 – 25.6
24	25.3 – 25.7
25	25.3 – 25.8
26	25.2 – 25.9
27	25.0 – 26.0
28	25.0 – 26.0
29	24.5 – 25.6
30	25.1 – 25.6
31	25.2 – 25.6
32	25.4 – 25.8
* Recorded using a min/max digital thermometer

 

Hatching success and fish larval survival during the fish early life stage test

Nominal Concentration (µg/L)	Vessel number	Number of eggs added at start of test		Number of hatched larvae		Percentage of hatched larvae (hatching success)		Number of larvae surviving at 28 days post-hatch		Percentage of larvae surviving at 28 days post-hatch (post-hatch survival)
		Individual vessels	Pooled vessels	Individual vessels	Pooled vessels	Individual vessels	Pooled vessels	Individual vessels	Pooled vessels	Individual vessels	Pooled vessels
Control	V1	20	80	20	80	100	100	16	70	80	88
	V2	20		20		100		19		95
	V3	20		20		100		17		85
	V4	20		20		100		18		90
Solvent control	V5	20	80	19	79	95	99	11	60	58	76
	V6	20		20		100		14		70
	V7	20		20		100		17		85
	V8	20		20		100		18		90
1.0	V9	20	80	20	80	100	100	19	72	95	90
	V10	20		20		100		18		90
	V11	20		20		100		17		85
	V12	20		20		100		18		90
3.2	V13	20	80	20	80	100	100	17	58	85	73
	V14	20		20		100		15		75
	V15	20		20		100		13		65
	V16	20		20		100		13		65
10	V17	20	80	20	80	100	100	8	46	40	58
	V18	20		20		100		14		70
	V19	20		20		100		16		80
	V20	20		20		100		8		40
32	V21	20	80	20	78	100	98	14	39	70	51
	V22	20		20		100		13		65
	V23	20		20		100		0		0
	V24	20		18		90		12		67
100	V25	20	80	*	59	*	98	*	7	*	12
	V26	20		19		95		0		0
	V27	20		20		100		3		15
	V28	20		20		100		4		20

* Eggs were lost possibly due to cleaning error. Not included in statistical analysis

 

Total length of fish (mm) at 28 days post-hatch

Fish Number	Nominal concentration (µg/L)
	Control				Solvent control				1.0
	Vessel number
	V1	V2	V3	V4	V5	V6	V7	V8	V9	V10	V11	V12
1	19.8	21.3	20.6	18.4	23.4	25.2	20.1	20.7	21.1	13.8	20.7	19.9
2	21.1	14.1	21.2	23.2	27.8	25.4	21.8	19.7	21.3	17.9	20.2	22.7
3	21.6	16.5	16.1	24.3	24.2	22.1	21.0	26.0	20.5	22.2	22.4	22.2
4	22.9	14.0	24.2	14.3	24.4	23.7	19.6	21.1	20.9	22.1	14.1	21.1
5	24.8	21.3	20.9	20.6	28.0	23.5	21.0	21.8	20.9	22.3	14.2	21.4
6	22.0	22.6	19.6	20.2	18.9	22.9	20.8	16.1	21.8	19.9	23.4	23.2
7	23.4	14.6	22.0	21.7	24.9	22.7	23.9	20.1	20.7	21.8	22.6	22.2
8	23.3	19.4	22.9	16.4	16.5	20.4	22.2	20.5	21.7	21.5	22.1	20.8
9	19.3	21.4	22.2	21.0	22.5	22.4	21.3	21.1	21.6	21.7	20.5	21.7
10	22.0	21.0	23.1	22.1	25.9	23.5	20.0	22.4	16.2	21.6	25.4	20.3
11	21.8	20.5	19.8	21.4	23.9	21.5	22.4	23.7	20.9	15.3	20.9	20.0
12	23.3	22.4	23.3	19.9	-	23.9	19.6	22.3	19.4	22.3	14.7	22.0
13	23.6	20.7	13.1	22.7	-	19.5	18.5	16.7	23.8	21.7	24.0	21.3
14	21.3	20.1	23.3	20.8	-	22.1	20.8	19.5	22.2	20.6	23.7	22.5
15	20.9	21.8	22.0	22.5	-	-	21.2	19.2	22.0	24.1	17.4	20.8
16	20.0	23.7	21.3	21.2	-	-	16.0	16.7	15.6	17.5	21.4	20.0
17	-	21.9	24.5	21.0	-	-	20.5	19.0	15.7	10.1	14.0	22.0
18	-	20.3	-	22.0	-	-	-	18.6	21.3	18.6	-	22.7
19	-	20.4	-	-	-	-	-	-	14.3	-	-	-
20	-	-	-	-	-	-	-	-	-	-	-	-
Mean	21.9	19.9	21.2	20.8	23.7	22.8	20.6	20.3	20.1	19.7	20.1	21.5
SD	1.5	2.9	2.9	2.4	3.4	1.6	1.7	2.5	2.6	3.6	3.8	1.0
Overall mean	20.9				21.6				20.4
Overall SD	2.6				2.7				3.0
-	Not applicable
SD	Standard deviation

Total length of fish (mm) at 28 days post-hatch (continued)

Fish Number	Nominal concentration (µg/L)
	3.2				10				32
	Vessel number
	V13	V14	V15	V16	V17	V18	V19	V20	V21	V22	V23*	V24
1	21.5	16.6	13.1	22.4	20.9	22.3	20.2	24.6	25.7	23.4	-	23.8
2	24.5	20.8	23.3	24.0	20.1	22.0	20.5	11.5	10.9	22.3	-	26.4
3	21.4	22.9	17.3	18.2	27.2	20.5	19.4	25.8	22.2	24.4	-	25.4
4	22.4	18.5	23.7	23.0	23.4	22.0	21.2	18.0	24.4	22.0	-	24.3
5	20.5	22.2	25.8	23.2	24.3	22.7	23.9	11.2	18.9	21.3	-	21.3
6	21.5	21.2	23.7	22.9	24.5	24.9	21.1	12.8	22.3	25.6	-	24.2
7	20.2	22.4	24.7	24.3	22.0	20.3	23.2	10.7	23.0	24.3	-	24.9
8	20.5	23.2	24.9	22.6	19.6	23.8	22.4	11.4	21.8	23.3	-	24.3
9	23.9	19.8	25.0	23.5	-	24.1	23.0	-	22.0	23.6	-	22.4
10	20.6	23.0	19.3	23.7	-	22.3	22.5	-	24.2	22.8	-	22.2
11	20.6	22.1	23.2	24.9	-	22.9	22.5	-	21.9	22.7	-	21.6
12	22.5	22.2	20.2	23.3	-	21.0	23.9	-	24.6	21.0	-	21.6
13	21.7	21.9	20.6	24.3	-	22.4	23.0	-	24.2	21.4	-	-
14	19.7	22.7	-	-	-	22.4	20.0	-	22.6	-	-	-
15	22.4	22.5	-	-	-	-	24.3	-	-	-	-	-
16	23.0	-	-	-	-	-	23.9	-	-	-	-	-
17	22.0	-	-	-	-	-	-	-	-	-	-	-
18	-	-	-	-	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-	-	-	-	-
Mean	21.7	21.5	21.9	23.1	22.8	22.4	22.2	15.8	22.1	22.9	-	23.5
SD	1.3	1.9	3.7	1.6	2.6	1.3	1.6	6.3	3.6	1.4	-	1.7
Overall mean	22.0				21.2				22.8
Overall SD	2.3				3.9				2.5
-	Not applicable
SD	Standard deviation
*	Eggs were lost possibly due to cleaning error. Not included in statistical analysis

Total length of fish (mm) at 28 days post-hatch (continued)

Fish Number	Nominal concentration (µg/L)
	100
	Vessel number
	V25	V26	V27	V28
1	-	-	26.5	24.9
2	-	-	27.5	26.0
3	-	-	9.9	26.1
4	-	-	-	27.4
5	-	-	-	-
6	-	-	-	-
7	-	-	-	-
8	-	-	-	-
9	-	-	-	-
10	-	-	-	-
11	-	-	-	-
12	-	-	-	-
13	-	-	-	-
14	-	-	-	-
15	-	-	-	-
16	-	-	-	-
17	-	-	-	-
18	-	-	-	-
19	-	-	-	-
20	-	-	-	-
Mean	-	-	21.3	26.1
SD	-	-	9.9	1.0
Overall mean	24.0
Overall SD	6.3
-	Not applicable
SD	Standard deviation

 

Wet weights of fish (mg) at 28 days post‑hatch

Fish Number	Nominal concentration (µg/L)
	Control				Solvent control				1.0
	Vessel number
	V1	V2	V3	V4	V5	V6	V7	V8	V9	V10	V11	V12
1	74.6	85.9	77.7	57.1	119.2	143.1	100.5	80.9	85.8	22.0	68.6	73.1
2	93.8	16.3	86.9	122.4	207.3	156.3	87.8	87.0	86.5	71.2	75.7	102.3
3	120.2	30.8	38.9	116.9	144.8	100.5	81.3	171.7	78.3	113.1	94.3	96.6
4	126.6	19.7	131.9	23.9	148.6	118.3	73.5	93.8	75.3	112.3	17.6	90.6
5	159.3	91.1	94.8	75.4	214.9	124.6	94.5	101.1	78.5	109.8	17.5	86.2
6	105.7	121.4	69.0	72.0	79.8	140.0	101.3	38.9	86.1	81.0	115.9	114.7
7	105.7	18.7	120.8	95.7	142.2	107.1	131.3	103.2	87.6	109.1	88.2	90.9
8	120.3	73.5	119.1	35.6	27.9	89.0	103.8	89.2	86.4	104.2	121.4	76.1
9	94.5	80.2	82.8	101.6	101.5	110.2	85.5	95.7	86.0	95.0	84.0	87.6
10	107.4	89.8	131.2	95.3	166.3	130.4	63.9	105.5	226.0	112.7	133.9	69.9
11	78.2	75.6	75.8	89.0	153.6	99.7	104.3	128.8	81.1	25.2	75.3	67.0
12	122.4	111.1	112.5	68.5	-	135.8	70.0	110.9	76.2	107.2	21.4	100.4
13	144.5	82.8	18.4	118.4	-	76.9	66.1	27.2	106.3	92.4	132.9	91.8
14	87.2	92.3	125.5	82.3	-	115.9	69.8	76.0	95.3	92.2	120.5	99.4
15	92.3	109.2	94.5	109.8	-	-	96.9	52.3	101.4	120.6	53.5	66.9
16	66.1	133.6	84.2	90.5	-	-	22.3	32.0	21.5	41.9	117.0	71.5
17	-	111.0	125.5	92.2	-	-	93.5	68.5	23.6	10.1	17.3	95.8
18	-	78.1	-	109.1	-	-	-	87.2	83.5	81.6	-	100.5
19	-	83.7	-	-	-	-	-	-	16.1	-	-	-
20	-	-	-	-	-	-	-	-	-	-	-	-
Mean	106.2	79.2	93.5	86.4	136.9	117.7	85.1	86.1	83.2	83.4	79.7	87.9
SD	25.3	34.7	32.3	27.4	54.0	22.3	23.7	35.2	43.1	35.2	41.9	14.1
Overall mean	90.7				102.5				83.6
Overall SD	31.3				39.5				34.9
-	Not applicable
SD	Standard deviation

Wet weights of fish (mg) at 28 days post‑hatch (continued)

Fish Number	Nominal concentration (µg/L)
	3.2				10				32
	Vessel number
	V13	V14	V15	V16	V17	V18	V19	V20	V21	V22	V23*	V24
1	92.2	41.3	15.4	105.7	71.8	103.4	75.5	194.2	137.7	127.0	-	125.6
2	132.4	73.2	108.8	126.9	78.2	96.2	80.5	11.3	8.4	102.9	-	161.6
3	87.2	104.6	34.7	53.2	221.2	84.1	63.1	195.6	104.2	135.4	-	150.1
4	105.7	53.6	120.0	116.9	123.2	85.1	90.1	49.0	131.8	116.7	-	141.4
5	78.0	106.5	135.3	119.2	150.2	95.9	112.7	10.5	59.0	98.3	-	85.8
6	81.5	86.9	112.7	128.5	138.5	134.3	82.3	12.6	107.6	165.7	-	147.0
7	71.5	91.9	138.2	129.0	98.7	86.5	103.4	10.7	111.1	140.8	-	139.9
8	69.4	107.3	139.8	108.6	67.1	110.5	96.3	9.5	95.7	125.6	-	137.1
9	124.9	58.7	154.1	107.3	-	134.2	95.5	-	102.3	125.8	-	115.6
10	80.8	84.4	66.0	112.2	-	95.0	81.7	-	147.4	114.1	-	107.5
11	75.9	96.7	112.1	144.0	-	214.4	111.2	-	86.5	109.7	-	93.9
12	100.3	87.1	67.0	126.4	-	81.1	107.5	-	141.0	92.2	-	102.0
13	87.7	95.1	80.4	138.0	-	102.0	124.0	-	123.8	92.6	-	-
14	77.1	100.1	-	-	-	92.6	60.3	-	111.1	-	-	-
15	92.8	99.9	-	-	-	-	145.8	-	-	-	-	-
16	106.5	-	-	-	-	-	123.9	-	-	-	-	-
17	86.7	-	-	-	-	-	-	-	-	-	-	-
18	-	-	-	-	-	-	-	-	-	-	-	-
19	-	-	-	-	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-	-	-	-	-
Mean	91.2	85.8	98.8	116.6	118.6	108.2	97.1	61.7	104.8	119.0	-	125.6
SD	17.8	20.4	42.8	22.4	51.8	34.8	23.3	83.3	36.4	21.0	-	24.3
Overall mean	97.2				98.1				115.9
Overall SD	28.5				48.7				29.0
-	Not applicable
SD	Standard deviation
*	Eggs were lost possibly due to cleaning error. Not included in statistical analysis

Wet weights of fish (mg) at 28 days post‑hatch (continued)

Fish Number		Nominal concentration (µg/L)
		100
		Vessel number
		V25		V26	V27	V28
1	-		-		212.3	160.2
2	-		-		236.7	213.1
3	-		-		6.9	190.5
4	-		-		-	234.2
5	-		-		-	-
6	-		-		-	-
7	-		-		-	-
8	-		-		-	-
9	-		-		-	-
10	-		-		-	-
11	-		-		-	-
12	-		-		-	-
13	-		-		-	-
14	-		-		-	-
15	-		-		-	-
16	-		-		-	-
17	-		-		-	-
18	-		-		-	-
19	-		-		-	-
20	-		-		-	-
Mean	-		-		152.0	199.5
SD	-		-		126.2	31.7
Overall mean	179.1
Overall SD	80.4
-	Not applicable
SD	Standard deviation

Validity criteria fulfilled:

yes

Conclusions:

The objective of the study was to define the lethal and sub-lethal effects of the test substance on the early developmental and juvenile stages of the freshwater fish species, Pimephales promelas, in accordance with OECD Chemical Testing Guideline No. 210.
No statistically significant differences were observed in terms of hatching success or final length and wet weight at any of the test concentrations employed in the test compared to the combined controls. Statistically significant differences were observed in terms of post-hatch survival at the 10, 32 and 100 µg/L test concentrations compared to the combined controls.
Given that the organisms were exposed to a mixture of both parent test substance and oxidation product, the results have been calculated based on nominal concentrations and geometric mean measured concentrations of the parent test substance.
The NOEC and LOEC values for hatching success were considered to be 100 and >100 µg/L, respectively, based on nominal concentrations (44 and >44 µg/L, respectively, based on geometric mean measured concentrations of the parent test substance).
The NOEC and LOEC values for post-hatch survival were considered to be 3.2 and >10 µg/L, respectively, based on nominal concentrations (2.1 and 13 µg/L, respectively, based on geometric mean measured concentrations of the parent test substance).
Taking both length and weight into consideration, the NOEC and LOEC values for growth were considered to be 100 and >100 µg/L, respectively, based on nominal concentrations (44 and >44 µg/L, respectively, based on geometric mean measured concentrations of the parent test substance).

Executive summary:

The objective of the study was to define the lethal and sub-lethal effects of the test substance on the early developmental and juvenile stages of the freshwater fish species Pimephales promelas, in accordance with OECD Chemical Testing Guideline No. 210.

 

Information supplied by the Sponsor gave an acute LC50 value for fathead minnow of 0.12 mg/L. Based on this information, the definitive test was conducted at nominal concentrations of 1.0, 3.2, 10, 32 and 100 µg/L. Control and solvent control groups were also included.

 

The test was conducted under flow-through conditions with continual renewal of the test media, using embryos produced by in-house brood fish which, on exposure, were at the early embryo/tail bud stage of development.

 

The analyte is highly unstable, being extremely sensitive to oxidation. The method used measured both the parent analyte, Lowinox® AH-25, and its oxidation product. Lowinox® AH-25 was quantified against matrix-matched standards. True quantification of the oxidation product was not possible since no primary standard was available. The peak area ratio of the parent to the oxidation product was reported during sample analysis to provide an indication of the relative amounts present.

 

Measured concentrations of the parent test substance showed variability throughout the duration of the test. This was due to the unstable nature of the test substance in the test media. The use of flow-through conditions and daily renewal of the solvent stock solutions assisted in maintaining relatively consistent concentrations in each test group. The peak area ratio of the parent to the oxidation product was also monitored during chemical analysis which indicated consistency throughout the duration of the test.

 

Given that the organisms were exposed to a mixture of both parent test substance and oxidation product, the results have been calculated based on nominal concentrations and geometric mean measured concentrations of the parent test substance. The geometric mean measured concentrations of parent test substance were calculated to be 2.1, 13, 31 and 44 µg/L for the 3.2, 10, 32 and 100 µg/L test concentrations, respectively. As analytical results were below the limit of quantification at all sampling occasions for the 1.0 µg/L test group, the this concentration was below the NOEC, a geometric mean measured concentration was not calculated. 

 

The mean hatching success of embryos in the control and solvent control groups was 100% and 99%, respectively, which was in excess of the validity criterion of ≥70%. The post-hatch survival in the control and solvent control groups was 88% and 76%, respectively, which was greater than the validity criterion of ≥75%. The test is therefore considered to be valid.

 

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

No statistically significant differences were observed in terms of hatching success or final wet weight and length at any of the test concentrations employed in the test compared to the combined controls. Significant differences were observed between the 10, 32 and 100 µg/L test concentrations and the combined controls in terms of post-hatch survival.

 

The NOEC and LOEC values for hatching success, post-hatch survival and growth (taking into account final fish length and wet weight) are summarised below:

Endpoint	Nominal concentration (µg/L)		Geometric mean measured concentration of parent compound (µg/L)
	NOEC	LOEC	NOEC	LOEC
Hatching success	100	>100	44	>44
Post-hatch survival	3.2	10	2.1*	13
Growth (Length)	100	>100	44	>44
Growth (Weight)	100	>100	44	>44

* Measured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution.

Description of key information

The NOEC and LOEC values for hatching success, post-hatch survival and growth (taking into account final fish length and wet weight) are summarised below:

Endpoint	Nominal concentration (µg/L)		Geometric mean measured concentration of parent compound (µg/L)
	NOEC	LOEC	NOEC	LOEC
Hatching success	100	>100	44	>44
Post-hatch survival	3.2	10	2.1*	13
Growth (Length)	100	>100	44	>44
Growth (Weight)	100	>100	44	>44

* Measured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

3.2 µg/L

Additional information

The objective of the study was to define the lethal and sub-lethal effects of the test substance on the early developmental and juvenile stages of the freshwater fish species Pimephales promelas, in accordance with OECD Chemical Testing Guideline No. 210.

 

Information supplied by the Sponsor gave an acute LC50 value for fathead minnow of 0.12 mg/L. Based on this information, the definitive test was conducted at nominal concentrations of 1.0, 3.2, 10, 32 and 100 µg/L. Control and solvent control groups were also included.

The test was conducted under flow-through conditions with continual renewal of the test media, using embryos produced by in-house brood fish which, on exposure, were at the early embryo/tail bud stage of development.

 

The analyte is highly unstable, being extremely sensitive to oxidation. The method used measured both the parent analyte, Lowinox® AH-25, and its oxidation product. Lowinox® AH-25 was quantified against matrix-matched standards. True quantification of the oxidation product was not possible since no primary standard was available. The peak area ratio of the parent to the oxidation product was reported during sample analysis to provide an indication of the relative amounts present.

Measured concentrations of the parent test substance showed variability throughout the duration of the test. This was due to the unstable nature of the test substance in the test media. The use of flow-through conditions and daily renewal of the solvent stock solutions assisted in maintaining relatively consistent concentrations in each test group. The peak area ratio of the parent to the oxidation product was also monitored during chemical analysis which indicated consistency throughout the duration of the test.

 

The mean hatching success of embryos in the control and solvent control groups was 100% and 99%, respectively, which was in excess of the validity criterion of ≥70%. The post-hatch survival in the control and solvent control groups was 88% and 76%, respectively, which was greater than the validity criterion of ≥75%. The test is therefore considered to be valid.

No statistically significant differences were observed in terms of hatching success or final wet weight and length at any of the test concentrations employed in the test compared to the combined controls. Significant differences were observed between the 10, 32 and 100 µg/L test concentrations and the combined controls in terms of post-hatch survival.

 

The NOEC and LOEC values for hatching success, post-hatch survival and growth (taking into account final fish length and wet weight) are summarised below:

Endpoint	Nominal concentration (µg/L)		Geometric mean measured concentration of parent compound (µg/L)
	NOEC	LOEC	NOEC	LOEC
Hatching success	100	>100	44	>44
Post-hatch survival	3.2	10	2.1*	13
Growth (Length)	100	>100	44	>44
Growth (Weight)	100	>100	44	>44

* Measured concentrations were less than the limit of quantification (LOQ), assessed as 5 µg/L. Results have been calculated using measured values or values of half LOQ were no test substance was detected. Result should be treated with caution.