Hydroquinone

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:

Experimental starting date is 21 April 2015 and Experimental completion date is 30 November 2015.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Singular samples for possible analysis were taken from all test concentrations and the control.

Sampling frequency : One day before the start of exposure to check the functioning of the system. At the start, after 7, 14, 21, 28 and 32 days of
exposure from one replicate per group changing systematically amongst replicates. In addition, samples were taken on day 28 from the stock solutions and the mixing vessels to check the functioning of the flow-through system.

Volume of samples : 2 mL

Storage of samples : Samples were freshly analysed on the day of sampling due to the expected instability in water.

Vehicle:

no

Details on test solutions:

PREPARATION OF STOCK SOLUTIONS
Stock solutions were prepared in purified water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges at nominal 10 and 100 mg/l. No treatment other than shaking was needed to fully dissolve the stock in Milli-Q water. The pH of the stock solutions was adjusted to pH 5 using Hydrochloric acid as to maintain the concentration in the stock stable. No correction was made for the purity/composition of the test item.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name ans strain: Fathead minnow (Pimephales promelas, Teleostei Cyprinidae) Rafinesque.
- Source: In house culture.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Ratio male/female: 1:2
- Spawning tank: The spawning tank is equipped with a substrate (pvc-tube), which enables collection of the fertilised eggs.
- Feeding brood stock: Frozen brine shrimp Nauplii and pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands).
- Time of fertilisation: Males and females are put together in spawning tanks and spawning starts the following day approximately 1 to 2 hours after lights have been switched on.

POST-HATCH FEEDING
Introduction egg: before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation)
Feeding : Embryonic phase: no feeding; Day 6-18: Brine shrimp Nauplii 24-hours old; Day 19-32: Brine shrimp Nauplii 48-hours old; Food was supplied ad libitum.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Post exposure observation period:

None

Hardness:

Total hardness was between 161 and 179 mg calcium carbonate per litre and thus above 140 mg/l asprescribed by the study plan. (Hardness was measured at the start and the end of the test in all test groups).

Test temperature:

The average temperature measured at weekly intervals in the controls and the treatment groups varied between 24.5 and 26.0°C. The temperature continuously measured in one of the replicates of the control was maintained between 24.2 and 25.8°C. Hence, temperature was within the range described in the study plan: 25 ± 1.5°C.

pH:

The pH varied between 7.3 and 7.8 during the test period, which was within the range described in the study plan (6.0-8.5). (pH was measured at the start of the test, at weekly intervals and at the end of the test in one replicate per group).

Dissolved oxygen:

Oxygen concentrations varied between 7.7 and 9.4 mg/l during the test period, which is > 60% of oxygen saturation at 25°C as prescribed by the study plan. (The dissolved oxygen was measured at the start of the test, at weekly intervals and at the end of the test in one replicate per group).

Salinity:

No measurement.

Nominal and measured concentrations:

Target concentrations : 4.6, 10, 22, 46 and 100 µg/l.
Measured concentrations are described in the section "details on results".

Details on test conditions:

SELECTION OF THE EXPOSURE REGIME
The test item is known to be unstable in water and therefore pre-test experiments were performed to determine and justify the exposure conditions. Moreover, both hydroquinone and the degradation product were monitored during the study. Flow-through conditions, meaning a continuous renewal of the test solutions, were selected to guarantee stable exposure concentrations. This has been checked during the range-finding test by
taking samples at three time points, i.e. on 2 and 1 day before initiation of the flow-through study to check the system and on the day of the start. As measured concentrations were in agreement with the target concentrations, it was decided to continue the testing using the system as is, without adaptation.

TEST SYSTEM
- Dosing system: Exact volumes of the test item stock solutions in Milli-Q water were dosed with syringes via a computer-controlled system consisting of five dispensers (Gilson). The dosed volumes of the stock entered a mixing flask separately from the dilution water supply (adjusted ISO-medium). The dilution water was supplied applying flow-meters at a constant rate of 7 litres per hour.
In the mixing flask the dosed volume and the dilution water were mixed under continuous stirring. Peristaltic pumps, set at a rate of 1 litre per hour, were used to divide the contents of the mixing vessels continuously and equally over four replicate test vessels containing the eggs/fish larvae/juvenile fish. The flow meters were calibrated before the start of the exposure and weekly thereafter. The whole system was checked twice daily.
- Test type: Flow-through, with continuous renewal of test media
- Test duration: 32 days
- Introduction egg: before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation)
- Test vessels: Stainless steel vessels (~1.7 l).
- Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
- Experimental design: The experiment (nominal day 0) started with 80 fresh and healthy fertilised fathead minnow eggs per test group. The fertilised eggs were randomly distributed and divided equally over four stainless steel vessels. Each vessel contained 20 eggs in 1.7 l test medium. The media were continuously renewed (flow-through).
- Light period: 16 h photo-period daily, between 555 and 592 lux.
- Feeding: Embryonic phase: no feeding; Day 6-18: Brine shrimp Nauplii 24-hours old; Day 19-32: Brine shrimp Nauplii 48-hours old; Food was supplied ad libitum.
- Euthanasia: At the end of the test the surviving larvae were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Salinity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Stage of embryonic development: Daily, from the beginning of the exposure
- Hatching and survival: Daily from nominal day 3, numbers of hatched larvae and dead embryos, larvae and juvenile fish were recorded. Dead embryos, larvae and juvenile fish were removed directly after recording.
- Criteria for death of Eggs: particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitating of protein, characterised by a white opaque appearance;
- Criteria for death of Embryos: Absence of body movement and /or absence of heartbeat;
- Criteria for death of Larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of the central nervous system and/or lack of reaction to mechanical stimulus.
- Abnormal appearance: Daily, abnormalities were recorded, e.g. hyperventilating, uncoordinated swimming, atypical quiescence and atypical swimming behaviour.
- Body weight: At the end of the test, all surviving fish were weighed on a replicate basis (blotted dry weight).
- Body length: At the end of the test, individual lengths from the surviving fish were measured.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY
See the section "any other information on materials and methods".

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 66 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

FIRST FINAL TEST
A first final study was performed with target concentrations of 2.2, 4.6, 10, 22 and 46 µg/l. This study was stopped after 22 days of exposure due to the fact that the validity criterion for post-hatch survival of larvae of at least 75% in the control was not met. After 22 days of exposure there appeared to be no significant effects in any of the Hydroquinone test groups including the target concentration of 46 µg/l as survival in these groups was equal or even higher than the observed control survival. It was consequently decided to repeat the study with a slightly higher test range of 4.6, 10, 22, 46 and 100 µg/l.

FINAL TEST
- Measured concentrations: An overview of the results of the analysis of the samples taken during the test period is presented below in Table 1. Analyses showed that measured concentrations were just below the target concentrations at the start and after 7 days of exposure with recoveries that generally ranged between 70 and 100%. The recoveries decreased as exposure progressed with recoveries dropping to 60-70% after 14 days and recoveries from day 21 ranged between approximately 35 and 60% relative to the target concentrations. The reason for the lower recoveries was not exactly known but there was a suspect that the increasing fish size in combination with the increasing food amount (live artemia) might have caused a decrease in actual test concentrations, e.g. by digestion or adsorption of test item from the water phase. The range tested based on the arithmetic mean measured concentrations was 2.5, 5.7, 14, 28 and 66 µg/l.
Concentrations of the degradation product p-benzoquinone were also measured. The contribution of the degradation product in solution at the highest target concentration of 100 µg/l was between 8 and 19% (i.e. relative to the measured Hydroquinone concentration).
- Embryonic survival, development and hatching: Table 2 shows the mean data for egg survival during the first 5 days of exposure for each of the
replicates. During the embryonic phase (1st four days) no significant differences were observed in numbers of embryos that were found dead in the controls and the various Hydroquinone test groups. Only two embryos died before hatching was complete; one embryo in the control and one embryo exposed to
28 µg/l. Mean survival of eggs in the control group was 99%. Hence, the validity criterion of >70% survival until hatching was complete was met. It was concluded that Hydroquinone did not affect survival of embryos up to and including a mean measured concentration of 66 µg/l.
Table 2 also includes the data of hatching starting on the fourth day and monitored until the fifth day. No differences were observed in the development of the embryos until hatching in either the control or treatments. Hatching started in the morning of day 4 and was complete in the afternoon of day 5. At the time points of recording, most embryos appeared to be at the same stage of development. It was concluded that Hydroquinone did not affect time of hatching or the hatching success up to and including a mean measured concentration of 66 µg/l.
- Larval survival and development: All hatched larvae in the control group survived the 32-day test period. Hence, the mean post-hatch larval survival for the control was 100% and thus the validity criterion for post-hatch survival of at least 75% was met. Mean survival rates in the different Hydroquinone concentrations were also high with 98, 94, 90, 94 and 86% for respectively 2.5, 5.7, 14, 28 and 66 µg/l. The survival rates were only slightly below the survival noted in the control group. Statistical analyses confirmed that larval survival was not significantly affected up to and including a mean measured concentration of 66 µg/l. Hence, the NOEC for post-hatch larval survival was 66 µg/l
Except for some minor incidental cases, no significant treatment related sub lethal effects were recorded in the Hydroquinone treated solutions.
- Effects on larval growth: Body lengths and body weights measured at the end of the test period are summarised in table 3 and Table 4 respectively. Mean length and mean weight of the fish exposed to the Hydroquinone treated test groups was comparable to that of the control group. Statistical analyses confirmed that the NOEC for growth (length and weight) equalled the highest test group of 66 µg/l.

DISCUSSION
The OECD Testing Guideline No 210 recommends that when the measured concentrations do not remain within 80-120% of the nominal concentration, the effect concentrations should be determined and expressed relative to the arithmetic mean concentration for flow-through tests.
At the highest target concentration of 100 µg/l, analyses showed that recoveries of hydroquinone decreased as exposure progressed with recoveries ranging from 35% to 96%. However, the degradation product p-benzoquinone can also induce effects. Hence the contribution of this degradation product to the test item concentration should be considered.
The contribution of the degradation product p-benzoquinone in solution varied from 6.4 to 42 % (i.e. relative to the measured Hydroquinone concentration). Globally it has been observed that the contribution of p-benzoquinone decreases as test item concentration increases. At the highest target concentration of 100 µg/l of hydroquinone, its contribution was between 8 and 19% (i.e. relative to the measured Hydroquinone concentration). Considering the measured concentrations of both hydroquinone and p-benzoquinone, the recoveries ranged from 45 to 107%, consequently still outside the accepted range of 80-120%. However, it should be highlighted that the analytical method development and validation demonstrated that there is a rapid interconversion between hydroquinone and p-benzoquinone in samples, and therefore the real concentration of both substances is uncertain.
Hence, the NOEC of Hydroquinone can be expressed both in arithmetic mean concentration as recommended by the OECD Testing Guideline No 210, and in nominal concentration to take into account the effects of the degradation product and the uncertainties of the analytical measurement.

CONCLUSION
The present study assessed the possible lethal and sub-lethal effects of Hydroquinone during the embryonic and early larval development of the fathead minnow. The results led to the following
conclusions for Hydroquinone:
1. Hydroquinone did not induce any significant, visible effects on the development of fathead minnow embryos at target concentrations up to and including 100 µg/l, corresponding to average measured concentrations up to and including 66 µg/l.
2. Hydroquinone did not significantly affect time of hatching or the hatching success at target concentrations up to and including 100 µg/l, corresponding to average measured concentrations up to and including 66 µg/l.
3. Hydroquinone did not affect survival, growth or development of the larvae during the whole test period at target concentrations up to and including 100 µg/l, corresponding to average measured concentrations up to and including 66 µg/l.
Hence, the NOEC of Hydroquinone for the early life stages of fish under the conditions of the present study is 100 µg/l expressed in target concentration and 66 µg/l expressed in average concentration measured in the highest tested group.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The following statistical procedures were used to determine the NOEC for embryonic and larval survival, body length and weight.
NOEC for larval survival:
• Differences between treatments and the control: Step-down Cochran-Armitage Test procedure (α=0.05, one-sided smaller).
NOEC for body length and body weight:
• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)
• Differences between treatments and the control: Body length; Dunnett’s multiple t-test procedure. Body Weight; Multiple Sequentially-rejective U-test after Bonferroni-Holm (α=0.05, one-sided smaller).

All analyses were performed with ToxRat Professional 3.0.0 (ToxRat Solutions® GmbH, Germany).

Table 1: Measured Hydroquinone concentrations vs target concentrations

Target (µg/l)	Measured Day 0 (µg/l)	Measured Day 7 (µg/l)	Measured Day 14 (µg/l)	Measured Day 21 (µg/l)	Measured Day 28 (µg/l)	Measured Day 32 (µg/l)	Mean measured (µg/l)
4.6	3.43	2.29	2.82	1.71	2.46	2.28	2.5
10	8.74	6.96	6.24	3.55	5.05	3.86	5.7
22	20.0	19.0	14.1	8.06	12.3	8.23	14
46	42.0	38.2	28.7	15.7	24.2	18.7	28
100	89.9	96.2	68.5	35.3	56.8	51.3	66

Table 2 : Egg survival and hatching success

Concentration Hydroquinone (µg/l)	(1)	(2)	(3)	(4)	(5)%	(4)	(5)%	(6) # survival after 114h	(6) % survival after 114h	(6) % survival after 114h
				90h	90h	114h	114h
Control	A	20	19	9	47	19	100	19	95	99
	B	20	20	9	45	20	100	20	100
	C	20	20	7	35	20	100	20	100
	D	20	20	3	15	20	100	20	100
2.5	A	20	20	6	30	20	100	20	100	100
	B	20	20	16	80	20	100	20	100
	C	20	20	14	70	20	100	20	100
	D	20	20	19	95	20	100	20	100
5.7	A	20	20	0	0	20	100	20	100	100
	B	20	20	0	0	20	100	20	100
	C	20	20	0	0	20	100	20	100
	D	20	20	18	90	20	100	20	100
14	A	20	20	17	85	20	100	20	100	100
	B	20	20	5	25	20	100	20	100
	C	20	20	2	10	20	100	20	100
	D	20	20	1	5	20	100	20	100
28	A	20	20	16	80	20	100	20	100	99
	B	20	20	2	10	20	100	20	100
	C	20	20	6	30	20	100	20	100
	D	20	19	2	11	19	100	19	95
66	A	20	20	15	75	20	100	20	100	100
	B	20	20	15	75	20	100	20	100
	C	20	20	19	95	20	100	20	100
	D	20	20	6	30	20	100	20	100
(1)  Vessel code. (2)  Number of eggs at start. (3)  Number of embryos at the start of hatching (day 4) (4)  Number larvae hatched after a defined period in hours. (5)  Percentage (%) hatched embryos relative to numbers present at the start of hatching (day 4). (6)  Number (#) and percentage (%) survival of embryos/larva of the total number eggs initially incubated.

Table 3: Individual body lengths (mm) of surviving larvae at the end of the test period

Code for larvae	Control						2.5 µg/l				5.7 µg/l
	A	B		C	D		A	B	C	D	A	B	C	D
Minimum	18.5	18.5		19.0	12.5		14.0	15.5	19.0	20.0	20.0	18.0	20.0	20.0
Maximum	25.0	24.5		25.0	24.0		25.0	25.0	25.0	24.0	24.0	24.0	24.0	25.0
Average	22.7	21.9		22.1	21.6		21.8	21.9	21.9	22.3	22.4	21.9	22.0	22.6
SD	1.48	1.51		1.63	2.57		2.35	1.92	1.73	0.96	1.18	1.55	1.24	1.36
CV	7%	7%		7%	12%		11%	9%	8%	4%	5%	7%	6%	6%
N	19	20		20	20		19	20	20	19	20	20	18	17
Code for larvae	14 µg/l					28 µg/l					66 µg/l
	A		B	C	D	A		B	C	D	A	B	C	D
Minimum	20.0		15.0	16.0	18.0	20.5		11.0	13.0	19.0	15.0	20.0	20.0	20.0
Maximum	25.0		25.0	25.0	25.0	25.0		24.0	26.0	25.0	25.0	24.5	24.0	25.0
Average	23.1		21.9	21.9	22.3	22.3		21.2	21.6	22.6	22.0	22.0	21.7	22.4
SD	1.45		2.11	2.13	1.80	1.24		3.21	2.70	1.79	2.95	1.27	1.16	1.24
CV	6%		10%	10%	8%	6%		15%	12%	8%	13%	6%	5%	6%
N	17		19	19	17	18		19	20	17	14	18	19	18

Table 4: Total body weights per replicate and mean weight per surviving larvae (mg wet weight) at the end of the test period

Concentration (µg/l)	Replicate	Group weight (mg)	Number of fish	Mean weight fish/replicate (mg)	Mean weight fish/group (mg)	SD (mg)	CV (%)
Control	A	1900.3	19	100.0	88.7	7.6	8.5
	B	1676.4	20	83.8
	C	1716.1	20	85.8
	D	1704.7	20	85.2
2.5	A	1685.0	19	88.7	87.1	1.6	1.9
	B	1718.3	20	85.9
	C	1709.6	20	85.5
	D	1676.4	19	88.2
5.7	A	1821.2	20	91.1	89.5	4.7	5.3
	B	1694.0	20	84.7
	C	1562.1	18	86.8
	D	1621.0	17	95.4
14	A	1723.6	17	101.4	92.4	6.3	6.8
	B	1663.4	19	87.5
	C	1685.4	19	88.7
	D	1565.1	17	92.1
28	A	1569.4	18	87.2	84.8	7.6	9.0
	B	1475.4	19	77.7
	C	1596.0	20	79.8
	D	1605.1	17	94.4
66	A	1289.4	14	92.1	89.9	2.7	3.0
	B	1590.0	18	88.3
	C	1648.7	19	86.8
	D	1659.6	18	92.2

Acceptability of the test

1) The dissolved oxygen concentration was maintained above 60% of the air saturation value throughout the test;

2) The water temperature did not differ by more than ± 1.5°C between test chambers between successive days at any time during the test, and was within the temperature ranges specified for the test species;

3) Exposure concentrations were analytically monitored throughout the test. Effect parameters were based on mean measured concentrations.

4) Overall survival of fertilised eggs in the controls was >70% until hatching was complete (99%). The overall survival of hatched larvae was >75% in the controls during the remaining test period (100%).

List of deviations

- study plan deviations: The procedure for sample analyses in the final study was based on project 509658 instead of project 507137 as included in the protocol. Evaluation: Additional effort was put in method development under a different project number to enable more sensitive measurements in the final study. The study integrity was not adversely affected by the deviation.

- standard operating procedures deviations: there were no deviations from standard operating procedures that affected the integrity of the study.

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the OECD TG 210. See section "any other information on results"

Conclusions:

The NOEC of Hydroquinone for the early life stages of fish under the conditions of the present study is 100 µg/l expressed in target concentration and 66 µg/l expressed in average concentration measured in the highest tested group.

Executive summary:

The purpose of the study was to estimate the possible lethal and sub-lethal effects of Hydroquinone during the embryonic and early larval development of the fathead minnow. This study was conducted according to the Fish early-life stage toxicity test under flow-through conditions and the GLP.

 

The study procedures were based on the OECD guidelines for Testing of Chemicals: Guideline No. 210, 2013. In addition, the procedures were designed to generally meet the test methods of the EPA Ecological Effects Test Guidelines, OPPTS 850.1400, Fish Early-Life Stage Test 'Public Draft', EPA 712-C-96- 121, April 1996.

 

The batch of Hydroquinone tested consisted of a white crystalline solid and the test item was completely soluble in test medium at the target concentrations tested. The test was performed using a flow-through system with target concentrations of 4.6, 10, 22, 46 and 100 µg/l, which were based on the results of a preceding range-finding test and a subsequent first invalid full study. An untreated control was also included.

 

In the flow-through system the dilution water was dosed separately from the test item stock solutions into mixing vessels. Stock solutions were prepared in purified water with a pH adjusted to 5. The dosed volumes of stock and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily.

 

The test was performed with four replicates containing 20 eggs per replicate for each concentration and control. The test started by placing fertilised eggs in stainless steel test vessels. During the embryonic and larval phases, the eggs/larvae were observed for survival and effects on development, appearance and swimming behaviour. At the end of the test (day 32), the surviving fish were measured and weighed.

 

Samples for chemical analysis of the actual Hydroquinone concentrations were taken one day before the start, at the start, at weekly intervals and at the end of the test. In addition, samples were taken at the same intervals to assess the concentration of the degradant p-benzoquinone in solution.

 

Analyses of samples taken from the target concentrations on a weekly basis showed that measured Hydroquinone concentrations were just below the target concentrations at the start and after 7 days of exposure with recoveries that generally ranged between 70 and 100%. The recoveries decreased as exposure progressed with recoveries dropping to 60-70% after 14 days and recoveries from day 21 ranged between approximately 35 and 60% relative to the target concentrations. The range tested based on the arithmetic mean measured concentrations was 2.5, 5.7, 14, 28 and 66 µg/l.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The present study assessed the possible lethal and sub-lethal effects of Hydroquinone during the embryonic and early larval development of the fathead minnow. The results led to the following conclusions for Hydroquinone:

 

1) Hydroquinone did not induce any significant, visible effects on the development of fathead minnow embryos at target concentrations up to and including 100 µg/l, corresponding to average measured concentrations up to and including 66 µg/l.

2) Hydroquinone did not significantly affect time of hatching or the hatching success at target concentrations up to and including 100 µg/l, corresponding to average measured concentrations up to and including 66 µg/l.

3) Hydroquinone did not affect survival, growth or development of the larvae during the whole test period at target concentrations up to and including 100 µg/l, corresponding to average measured concentrations up to and including 66 µg/l.

 

Hence, the NOEC of Hydroquinone for the early life stages of fish under the conditions of the present study is 100 µg/l expressed in target concentration and 66 µg/l expressed in average concentration measured in the highest tested group.

Description of key information

A study performed according to the OECD TG 210 and under GLP is available. No effect on the reproduction were observed up to and including the highest tested concentration. The NOEC is 100 µg/l expressed in target concentration and 66 µg/l expressed in average concentration measured in the highest tested group.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

>= 66 µg/L

Additional information

One reliable key study is available for this endpoint. The purpose of this study was to estimate the possible lethal and sub-lethal effects of Hydroquinone during the embryonic and early larval development of the fathead minnow. This study was conducted according to the Fish early-life stage toxicity test under flow-through conditions and the GLP.

 

The study procedures were based on the OECD guidelines for Testing of Chemicals: Guideline No. 210, 2013. In addition, the procedures were designed to generally meet the test methods of the EPA Ecological Effects Test Guidelines, OPPTS 850.1400, Fish Early-Life Stage Test 'Public Draft', EPA 712-C-96- 121, April 1996.

 

The batch of Hydroquinone tested consisted of a white crystalline solid and the test item was completely soluble in test medium at the target concentrations tested. The test was performed using a flow-through system with target concentrations of 4.6, 10, 22, 46 and 100 µg/l, which were based on the results of a preceding range-finding test and a subsequent first invalid full study. An untreated control was also included.

 

In the flow-through system the dilution water was dosed separately from the test item stock solutions into mixing vessels. Stock solutions were prepared in purified water with a pH adjusted to 5. The dosed volumes of stock and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily.

 

The test was performed with four replicates containing 20 eggs per replicate for each concentration and control. The test started by placing fertilised eggs in stainless steel test vessels. During the embryonic and larval phases, the eggs/larvae were observed for survival and effects on development, appearance and swimming behaviour. At the end of the test (day 32), the surviving fish were measured and weighed.

 

Samples for chemical analysis of the actual Hydroquinone concentrations were taken one day before the start, at the start, at weekly intervals and at the end of the test. In addition, samples were taken at the same intervals to assess the concentration of the degradant p-benzoquinone in solution.

 

Analyses of samples taken from the target concentrations on a weekly basis showed that measured Hydroquinone concentrations were just below the target concentrations at the start and after 7 days of exposure with recoveries that generally ranged between 70 and 100%. The recoveries decreased as exposure progressed with recoveries dropping to 60-70% after 14 days and recoveries from day 21 ranged between approximately 35 and 60% relative to the target concentrations. The range tested based on the arithmetic mean measured concentrations was 2.5, 5.7, 14, 28 and 66 µg/l.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The present study assessed the possible lethal and sub-lethal effects of Hydroquinone during the embryonic and early larval development of the fathead minnow. The results led to the following conclusions for Hydroquinone:

 

1) Hydroquinone did not induce any significant, visible effects on the development of fathead minnow embryos at target concentrations up to and including 100 µg/l, corresponding to average measured concentrations up to and including 66 µg/l.

2) Hydroquinone did not significantly affect time of hatching or the hatching success at target concentrations up to and including 100 µg/l, corresponding to average measured concentrations up to and including 66 µg/l.

3) Hydroquinone did not affect survival, growth or development of the larvae during the whole test period at target concentrations up to and including 100 µg/l, corresponding to average measured concentrations up to and including 66 µg/l.

 

Hence, the NOEC of Hydroquinone for the early life stages of fish under the conditions of the present study is 100 µg/l expressed in target concentration and 66 µg/l expressed in average concentration measured in the highest tested group.

The OECD Testing Guideline No 210 recommends that when the measured concentrations do not remain within 80-120% of the nominal concentration, the effect concentrations should be determined and expressed relative to the arithmetic mean concentration for flow-through tests.

At the highest target concentration of 100 µg/l, analyses showed that recoveries of hydroquinone decreased as exposure progressed with recoveries ranging from 35% to 96%. However, the degradation product p-benzoquinone can also induce effects. Hence the contribution of this degradation product to the test item concentration should be considered.

The contribution of the degradation product p-benzoquinone in solution varied from 6.4 to 42 % (i.e. relative to the measured Hydroquinone concentration). Globally it has been observed that the contribution of p-benzoquinone decreases as test item concentration increases. At the highest target concentration of 100 µg/l of hydroquinone, its contribution was between 8 and 19% (i.e. relative to the measured Hydroquinone concentration). Considering the measured concentrations of both hydroquinone and p-benzoquinone, the recoveries ranged from 45 to 107%, consequently still outside the accepted range of 80-120%. However, it should be highlighted that the analytical method development and validation demonstrated that there is a rapid interconversion between hydroquinone and p-benzoquinone in samples, and therefore the real concentration of both substances is uncertain.

Hence, the NOEC of Hydroquinone can be expressed both in arithmetic mean concentration as recommended by the OECD Testing Guideline No 210, and in nominal concentration to take into account the effects of the degradation product and the uncertainties of the analytical measurement.