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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 June 2022 - 29 August 2022
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:
2013
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions (see Appendix 10 attached).
Qualifier:
according to guideline
Guideline:
other: OECD 23 Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals
Version / remarks:
8 February 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Omnirad 184, Hydroxycyclohexyl phenyl ketone
Physical Description: Fine to coarse white powder
Storage Conditions: At room temperature protected from light

Additional information
Test Facility test item number: 212711/A
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum foil
Chemical name (IUPAC, synonym or trade name): Hydroxycyclohexyl phenyl ketone
CAS number: 947-19-3
EC number: 213-426-9
Solubility in vehicle:
• Water 0.442 g/L @ 23oC
• Other: Not indicated
Stability in vehicle:
• Water Not indicated
• Other: Not indicated
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations, the controls and from stock solutions used for dosing. See the appended Analytical Report for the method of
analysis.

Frequency:
All test concentrations and controls: one day before the start of exposure to check the functioning of the system. At the exposure start (day 0) and at weekly intervals (i.e. on day 7, 14, 21, and 28 of exposure).

Stock solutions used for dosing:
- Highest stock concentration on days -3, -2 and -1 before the start of exposure, to check for test material stability in the solvent.
- All stock concentrations: one day before the start of exposure to check the functioning of the system. At the exposure start (day 0) and at weekly intervals (i.e. on day 7, 14, 21, and 28) of exposure.

Volume: 1.0 mL
Storage Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
Additionally, reserve samples of 1.0 mL were taken from all test solutions and stocks and stored in a freezer (set to maintain -20°C) for possible analysis.
Vehicle:
yes
Remarks:
DMSO
Details on test solutions:
Preparation of Stock and Test Solutions:
The batch of Omnirad 184, Hydroxycyclohexyl phenyl ketone tested was a fine to coarse white powder. No correction was made for the purity/composition of the test material.
The standard test procedures required generation of test solutions, which should contain completely dissolved test material concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system was prevented.

Range-Finding Test:
Aqueous stock solutions were prepared four times at a concentration of 100 mg/L applying vortexing followed by an overnight period of magnetic stirring to ensure complete dissolution. The obtained solution was used as highest test group. Lower test concentrations were prepared by dilution in test medium. All test solutions were clear and colourless at the end of the preparation procedure.
Any residual volumes were discarded.

Final Test:
Stock solutions of 100 mg/mL were prepared in dimethylsulphoxide (DMSO; Merck, Darmstadt, Germany) three times a week. Lower stock concentrations, i.e. 46, 22, 10 and 4.6 mg/mL were prepared by diluting the highest stock with DMSO. All stock solutions were clear and colourless. The stock solutions were dosed to test medium by means of a computer-controlled dosing system as described below.
Any residual volumes were discarded.
Test organisms (species):
Pimephales promelas
Details on test organisms:
Species: Fathead minnow (Pimephales promelas, Teleostei Cyprinidae) Rafinesque.
Reason for selection: This system has been selected as an internationally accepted species.
Source: In-house culture.

Holding of the Brood Stock
Holding medium: Test medium.
Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, temperature was measured before transferring the parental fish to the breeding system.
Water quality parameters: Were maintained within the optimum limits of the selected fish species.
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/or pelleted fish food (SDS 400, 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.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Post exposure observation period:
No
Hardness:
CaCO3: 180 mg/L.
Test temperature:
25 ± 1.5°C
pH:
Between 6.0 and 8.5
Dissolved oxygen:
≥ 4.9 mg/L (i.e. 60% of air saturation value at 25°C)
Salinity:
Not applicable.
Conductivity:
Not reported.
Nominal and measured concentrations:
Range-finding test:
Nominal: 1.0, 10 & 100 mg/l
Samples taken from the freshly prepared solutions showed that measured concentrations were generally in agreement with the nominal concentrations, i.e. were at 102 to 108% relative to the nominal values, with one exception for the highest test concentration on day 0 which was only 27% relative to nominal. The concentrations measured in the aged solutions were relatively stable and ranged from 61 to 97% relative to nominal after 24-, 48- and 72-hour intervals.

Main test:
Nominal: 0.46, 1.0, 2.2, 4.6 & 10 mg/l
Measured: 0.52, 1.1, 2.2, 4.6, 10 mg/l
Details on test conditions:
Test type: Flow-through, with continuous renewal of test media.
Introduction egg: Before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation).
Test vessels: Stainless steel vessels (~1.7 L).
Test medium: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
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 photoperiod daily.
Feeding Embryonic phase: no feeding.
Larvae and juvenile fish: Brine shrimp Nauplii 24 or 48 hours old. Food was supplied ad libitum.
Reference substance (positive control):
no
Remarks:
No positive control
Duration:
32 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
32 d
Dose descriptor:
EC10
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
32 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
32 d
Dose descriptor:
EC10
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
32 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
32 d
Dose descriptor:
EC10
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
5 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Embryonic survival
Duration:
5 d
Dose descriptor:
EC10
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Embryonic survival
Duration:
5 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Embryonic survival
Duration:
5 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Embryonic survival
Details on results:
Range-Finding Test
The results of the semi-static range-finding test are presented in Table 2. Hatching started on day 4 and was complete on day 7 for all tested groups. No test material related mortality or significant sublethal effects were observed at the lowest two test concentrations or the control during the 8-day test period. However, at the highest test concentration (i.e. 100 mg/L), none of the introduced eggs hatched and all died within 6 days of exposure. Hence, larval development and time of hatching was only affected in the highest tested concentration compared to the control solution.
Samples taken from the freshly prepared solutions showed that measured concentrations were generally in agreement with the nominal concentrations, i.e. were at 102 to 108% relative to the nominal values, with one exception for the highest test concentration on day 0 which was only 27% relative to nominal. The concentrations measured in the aged solutions were relatively stable and ranged from 61 to 97% relative to nominal after 24-, 48- and 72-hour intervals. The results of the analysis of the samples taken during the range-finding test are described in Table 2 of the appended Analytical Report.
All test conditions were maintained within the limits prescribed by the study plan.
Based on the results of this preliminary test, it was decided to test a range of target concentrations between 0.46 and 10 mg/L in the main flow-through study.

Early Life Stage Test
Measured Concentrations
The results of the analysis of the samples taken during the test period are described in Table 3 (stock solutions) and Table 4 (test solutions) of the appended Analytical Report.
Stock stability was tested on a 100 mg/mL stock before exposure started showing that the stock was stable for at least three days at room temperature whilst stirring. Stock solution renewal frequency was based on this information.
Analyses of samples taken from the stocks and target concentrations on a weekly basis showed that measured concentrations were in agreement with the targets, ranging between approximately 88 to 110% relative to the respective targets for the stocks, and 96 to 119% relative to the respective targets for the test solutions.
The calculated average exposure concentrations are presented in Table 3. The mean measured concentrations were at 99-112% of the targets. The coefficient of variation was between 3 and 6% indicating stable dosing throughout the exposure period.
The effect parameters were based on the mean measured concentrations.

Embryonic Survival
Figure 1 shows the mean data for hatchability (egg survival) during the first 5 days of exposure for each of the test groups. Appendix 1 lists the survival data per vessel throughout the test period and Table 4 summarises the effects at the end of the hatching. Determination of the NOEC is presented in Appendix 4.
Hatching commenced on day 3 and was near to complete on day 5 of exposure. After day 5, three eggs remained unhatched at the 2.2, 4.6 and 10 mg/L groups, but were observed dead on days 7, 8 and 7, respectively.
The overall survival of embryos at the end of hatching was 99 and 98% for the blank- and solvent control, respectively. Hence, the validity criterion of >70% survival until hatching was complete was met. Because no statistically significant difference was observed between control treatments, both blank- and solvent control were pooled.
No statistically significant differences were observed at any of the concentrations tested when compared to the pooled control. Hence, the NOEC for embryonic survival/hatching success was 10 mg/L.

Larval Survival and Development
Figure 2 shows a graphical presentation of the larval survival per test group during the post-fertilization period up to day 32 of exposure. Appendix 1 lists the survival data per vessel throughout the test period and Table 5 summarises the effects at the end of exposure. Determination of the NOEC is presented in Appendix 5.
The mean post-hatch larval survival was 95% for the blank control and 91% for the solvent control. Hence, the validity criterion for post-hatch survival of at least 75% was met. Because no statistically significant difference was observed between control treatments, both blank and solvent control were pooled.
Statistical analyses confirmed that larval survival was not significantly affected up to and including 10 mg/L (NOEC) when compared to the pooled control.
No significant effects were observed in the control treatments or in any of the test concentrations. The few cases of developmental defects in the control groups and the test concentrations were considered incidental and unrelated to the applied treatment as the severity or numbers of fish affected did not increase with increased treatment concentration and were comparable to the control treatment observations. An overview of all symptoms is presented in Appendix 1.

Effects on Larval Growth
Body lengths and body weights measured at the end of the test period are presented in Appendix 2, Table 18 and Appendix 3, Table 19, respectively. Graphical presentations of body lengths and weights in different test groups are shown in Figure 3 and Figure 4, respectively. Table 6 and Table 7 summarise the effects at the end of exposure. Determination of the NOEC is presented in Appendix 6 for body weight and Appendix 7 for body length.
Mean body weight of the larvae at the end of the test was 74.6 and 84.1 mg in the blank control and the solvent control, respectively. Mean body length of the larvae at the end of the test was 21.0 to 21.7 mm in the blank control and the solvent control, respectively. Because no statistically significant difference was observed for both body weight and length between control treatments, the blank and solvent control were pooled.
No statistically significant differences for body weight were observed at any of the concentrations tested when compared to the pooled control. Omnirad 184, Hydroxycyclohexyl phenyl ketone caused a statistically significant reduction (5.2% compared to the pooled control) in body length in the highest concentration. Hence, the NOEC for growth was 4.6 mg/L based on body length.

Experimental Conditions
The experimental conditions are summarized in Table 9. The individual results are presented in:
• pH: Appendix 8, Table 20. The pH was within the range described in the study plan (6.0-8.5).
• Oxygen concentration: Appendix 8, Table 21. Oxygen concentrations were > 60% (i.e. >4.9 mg/L) of oxygen saturation at 25°C as prescribed by the study plan.
• Temperature: Appendix 8, Table 22. The average temperature measured at weekly intervals in the controls and the treatment groups was within the range described in the study plan: 25 ± 1.5°C. The average temperature continuously measured in one of the replicates of the control was within the range described in the study plan: 25 ± 1.5°C.
• Hardness: Appendix 8, Table 23. Total hardness was >140 mg/L, as prescribed by the study plan.
• The concentration of TOC in a sample taken from the test medium was below the detection limit of the device and as such complied with the value recommended by the guideline (i.e. <2 mg TOC/L).


Table 2: Survival of Fish Embryos and Larvae During the Range-Finding Test






















































































































Omnirad 184,
Hydroxycyclohexyl phenyl ketone
Nominal (mg/L)
No. of eggsNumber of survivors (at nominal day)Total survival (%)
Day 012345678
Control101010101010101010100
101010101010101010
110101010101010101095
1099996999
10101010101010101010100
101010101010101010
100101010101070000
1097660000

 


Table 3: Nominal and Mean Measured Concentrations





































































Omnirad 184, Hydroxycyclohexyl phenyl ketoneMeasured concentration (mg/L) at dayMean exposure conc. (mg/L)%CV1
Nominal conc. (mg/L)07142128
0.460.4820.5470.5450.5070.5010.526
11.011.141.111.031.041.15
2.22.132.322.262.132.192.24
4.64.44.784.614.44.654.64
109.8610.410.29.629.88103

1 Coefficient of variation


 


Table 4: Percentage of Embryonic Survival at the End of Hatching (Day 5)






















































Omnirad 184,
Mean measured conc. (mg/L)
Hydroxycyclohexyl phenyl ketone 
Total IntroducedHatchedNot Hatched% Hatched
Pooled control160157398
0.528079199
1.18075594
2.28077396
4.68078298
108079199

 


Table 5: Post-Hatch Mortality at the End of Exposure (Day 32)






















































Omnirad 184,
Hydroxycyclohexyl phenyl ketone
Mean measured conc. (mg/L)
Total1 introducedSurvivedDead% Post-hatch mortality
Pooled control157146117
0.52797456.3
1.1757234
2.2777167.8
4.6787800
10797278.9

1 Total number of hatched larvae


 


Table 6: Mean Fresh Weight (mg) After 32 Days of Exposure






















































Omnirad 184,
Hydroxycyclohexyl phenyl ketone
Mean measured conc. (mg/L)
MeanStd. Dev.n%Reduction#
Pooled control79.38.88 
0.5284.72.231-6.7
1.189.38.84-12.6
2.284.76.84-6.8
4.680.16.24-1
1078.38.141.3

# Negative numbers indicate increase instead of decrease; n: number of replicates
1 One fish from replicate D of the solvent control group was accidentally left in the net when the fish of this replicate were collected for measuring body weight. As a result, this fish was included in the measurements taken from replicate A of the 0.52 mg/L group. Therefore, it was decided to exclude replicate A of the 0.52 mg/L group from the statistical analysis for body weight (see also Deviations in Appendix 10).


 


Table 7: Mean Body Length (mm) After 32 Days of Exposure






















































Omnirad 184,
Hydroxycyclohexyl phenyl ketone
Mean measured conc. (mg/L)
MeanStd. Dev.n%Reduction#
Pooled control21.320.6298 
0.5221.450.48431-1
1.121.640.4774-1.5
2.221.780.434-2.1
4.621.070.48141.2
1020.210.5274   5.2*

# Negative numbers indicate increase instead of decrease; n: number of replicates
1 One fish from replicate D of the solvent control group was accidentally left in the net when the fish of this replicate were collected for measuring body weight. As a result, this fish was included in the measurements taken from replicate A of the 0.52 mg/L group. Therefore, it was decided to exclude replicate A of the 0.52 mg/L group from the statistical analysis for body weight (see also Deviations in Appendix 10).
* Effect was statistically significant when compared to the pooled control.


 


Table 9: Summary of Experimental Conditions










































ConditionRange
pH7.2 – 7.8
Temperature measured in test vessels (°C)24.7 – 25.8
Temperature measured continuously in control vessel (°C)24.9 – 26.3
Dissolved oxygen concentration (mg O2/L)6.4 – 9.8
Hardness (mg CaCO3/L)179 - 196
Total Dissolved Organic Carbon content (mg TOC/L)<LOQ
Light intensity (Lux)Start test550 - 590
End test554 - 585

LOQ: Limit of quantification was 1 mg/L

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the present study assessed the possible lethal and sub-lethal effects of Omnirad 184, Hydroxycyclohexyl phenyl ketone during the embryonic and early larval development of the fathead minnow. The results led to the following conclusions for Omnirad 184, Hydroxycyclohexyl phenyl ketone:
• Omnirad 184, Hydroxycyclohexyl phenyl ketone did not affect the hatching success (embryonic survival) at mean measured concentrations up to and including 10 mg/L (NOEC). Hence the LOEC was >10 mg/L.
• Omnirad 184, Hydroxycyclohexyl phenyl ketone did not affect the survival of larvae at mean measured concentrations up to and including 10 mg/L (NOEC). Hence the LOEC was >10 mg/L.
• The test material had a significant effect on the growth of the exposed larvae. Body length was significantly affected at a mean measured concentration of 10 mg/L (LOEC), while body weight was not significantly affected at the end of the exposure. The NOEC was 10 and 4.6 mg/L for weight and length, respectively.
The overall NOEC of Omnirad 184, Hydroxycyclohexyl phenyl ketone for the early life stages of fish is 4.6 mg/L and the overall LOEC is 10 mg/L.
Executive summary:

The objective of the study was to define the lethal and sub-lethal effects of Omnirad 184, Hydroxycyclohexyl phenyl ketone on the early life stages of fish in a flow-through system. For this objective, the early-life stages of fathead minnow were exposed to a range of concentrations of the test material dissolved in water for a total period of 32 days. Lethal and sub-lethal effects were assessed and compared with control values to determine the various effect concentrations.
A full test was performed based on the results of a semi-static range-finding test. The study met the acceptability criteria prescribed by the study plan and was considered valid. Test conditions and results of the final test are presented in the table below:





































































































Test Material:Omnirad 184, Hydroxycyclohexyl phenyl ketone
Appearance:Fine to coarse white powder
Purity:99.70%
Correction factor:No correction factor required
Preparation of test solutions:Nominal concentrations (mg/L), individually dosed with stock solutions in DMSO
Specific procedures:Use of amber glassware for stocks and light conditions reduced to the lower end of the optimal range.
Experimental Set-up: 
Guidelines: OECD TG 210 and OECD GD 23 
Exposure design:Flow-through 
Test concentrations: 0.46, 1.0, 2.2, 4.6 and 10 mg/L (nominal)
Controls:Blank control and solvent control
Stock solutions:4.6, 10, 22, 46 and 100 mg/mL in DMSO
Number of replicates: Four replicates per test group, each containing 20 eggs at the start of the test
Sampling for analysis:One day before start, at the start and weekly thereafter. 
Experimental conditions: 
pH, Temperature, Dissolved Oxygen:Within the ranges specified in OECD TG 210
Light regime:A daily photoperiod of 16 hours
Results: 
Actual exposure concentrations:0.52, 1.1, 2.2, 4.6 and 10 mg/L (mean measured)
Comparison of control treatments:Statistical comparisons showed that there was no significant difference between any of the endpoints in the blank- and the solvent control. The use of the solvent was concluded not to affect the obtained results. Subsequently, the pooled control was used to test the potential effects of test material exposure.
Hatching success (Embryonic survival):No statistically significant differences were observed at any of the concentrations tested when compared to the pooled control.
Post-hatch survival (Larval survival):No statistically significant differences were observed at any of the concentrations tested when compared to the pooled control.
Growth:Omnirad 184, Hydroxycyclohexyl phenyl ketone caused a statistically significant reduction in body length. At the highest concentration, the reduction of length reached 5.2%. There were no statistically significant differences in body weight between the pooled control and the different test material treatments.
Other effects:Only a few incidences of developmental defects were observed in the control groups and the test concentrations during the exposure period. The severity and incidence of abnormalities was not dose related.

The effect parameters obtained in this study (based on mean measured concentrations) are summarized in the table below.








































Parameter LOECNOECEC10,50
Embryonic survivalValue1>1010>10
Post-hatch survivalValue2>1010>10
Growth (weight)Value2>1010>10
Growth (length)Value2104.6>10

1 for day 5 of exposure.


2 for day 32 of exposure.


The overall NOEC of Omnirad 184, Hydroxycyclohexyl phenyl ketone for the early life stages of fish is 4.6 mg/L and the overall LOEC is 10 mg/L.

Description of key information

The overall NOEC of Omnirad 184, Hydroxycyclohexyl phenyl ketone for the early life stages of fish is 4.6 mg/L and the overall LOEC is 10 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
4.6 mg/L

Additional information