Hydroxycyclohexyl phenyl ketone

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 October 2022 - 28 October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name: Hydroxycyclohexyl phenyl ketone
EC number: 213- 426-9
CAS number: 947-19-3
Description: White fine to coarse powder

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the controls.
The method of analysis is described in the Analytical Phase Report (Appendix 9).

Sampling:
Frequency
1. At the start of the test and weekly thereafter from the freshly prepared solutions (Day 0, 7, 14, 20).
2. At the first renewal (Day 2) and weekly thereafter from the 24-hour old solutions (Day 1, 8, 15, 21).

Volume - 1 mL per duplicate

Storage - Samples were stored in a freezer (set to maintain -20°C) until shipment to the Test Site #1 for analysis
 
At the end of the renewal periods, the replicates were pooled at each concentration before sampling and duplicate samples (Set #1 and Set #2; 2 x 1 mL in an amber glass vial of 4 mL) were taken from the test solutions on each occasion, which samples then were frozen and stored in freezer (approx. -20°C) until shipment. Thereafter, Set #1 samples were sent to the Test Site for analysis and Set #2 samples were retained frozen in freezer (approx. -20°C) as a back-up at the Test Facility.

Vehicle:

no

Details on test solutions:

Reconstituted water (Elendt M4 media, according to OECD 211 and 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.

Preparation of test solutions started with a loading rate of 100 mg/L applying a 24-hour period of magnetic stirring to accelerate dissolution of the test item in medium. The aqueous Saturated Solution (SS) was collected and filtered through a 0.45 µm membrane filter (Supplier: Merck KGaA; batch number: MP213802G2). The test solutions were clear and colourless at the end of the preparation procedure.

The test solution was prepared just before introduction of the Daphnia (start of the treatment).

The lower concentrations were prepared by appropriate dilution of this stock solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species and strain: Daphnia magna (Straus)
Source: Hungarian University of Agriculture and Life Sciences, Department of Aquaculture 2100 Gödöllő, Páter Károly u. 1. – Hungary

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

21 days

Hardness:

The reconstituted water (ISO medium) had a total hardness of above 140 mg/L (as CaCO3) (between 242 – 281 mg/L).

Test temperature:

The water temperature was measured daily in one control test vessel and weekly in one replicate vessel of the test medium. The test temperature was in the range of 19.6 - 22.0°C in the control vessels and in the range of 19.4 – 22.0°C in the test concentration vessels. The additionally measured temperature in the climate chamber was between 19.3 – 22.6°C.

pH:

The pH was measured at the start of the test (Day 0), on Day 1, 4, 5, 12, 13 and at the end of the test (day 20, 21) in the control and in one test vessel of the test item concentration and was in the range of 6.96 – 8.76.

Dissolved oxygen:

The dissolved oxygen concentration was measured in the control as well as in the test concentration level (in one test vessel) at the start of the test (Day 0), on Day 1, 4, 5, 12, 13 and at the end of the test (day 20, 21) and was in the range of 7.7 – 8.9 mg/L.

Salinity:

N/A - freshwater

Conductivity:

N/A - freshwater

Nominal and measured concentrations:

Range finding study: 100 mg/L

Definitive test 1
(nominal and geometric mean measured) 6.25, 12.5, 25, 50 and 100 mg/L

Definitive test 2:
The geometric mean measured concentrations of test item were the same as the nominal concentrations; 0.03, 0.1, 0.3, 1 and 3 mg/L.

Details on test conditions:

Number of animals: There were 10 animals at each group (test item concentrations and untreated control).
Loading: Parent animals were maintained individually, one per test vessel, with ~80 ml of test solution in each vessel.
Age of animals: Less than 24 h old at the beginning of the test and not first brood progeny.
Acclimatization: To avoid the necessity of adaptation prior to the test, the water used in the test was similar to the culture conditions (water temperature, background colour, light intensities).
Offspring: Produced by the parental animals in the course of this test. When the parental animals are about seven days old, the first young Daphnia emerge from the brood pouch, thereafter a new batch appears every two to three days. These batches are called "broods" of the F1 (filial 1) generation.
Holding water: Same water was used as test water (see section 6.3.).
Food and Feeding: The Daphnia were fed daily with concentrated algal suspension of Raphidocelis subcapitata.

The supplied diet was based on the amount of organic carbon (C) provided to each parent animal. The constant ration level was 0.15 mg C/Daphnia/day during the test.
The cell number of algal-suspension fed was counted before the test. The total organic carbon content (TOC) of algal-suspension fed was based on the results of the last annual nomographical TOC measurement (see Appendix 6).

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.29 mg/L

95% CI:

>= 0.1 - <= 0.89

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.25 mg/L

95% CI:

>= 0.04 - <= 0.5

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.19 mg/L

95% CI:

>= 0.9 - <= 1.57

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth

Key result

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

0.42 mg/L

95% CI:

>= 0.14 - <= 1.29

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

0.45 mg/L

95% CI:

>= 0.13 - <= 0.84

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

2.09 mg/L

95% CI:

>= 1.53 - <= 2.88

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.83 mg/L

95% CI:

>= 0.2 - <= 3.25

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1.38 mg/L

95% CI:

>= 0.73 - <= 3.96

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

6.14 mg/L

95% CI:

>= 3.51 - <= 10.47

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth

Details on results:

Validity of the study
There was no mortality in the control group. The mean number of live offspring per parent animal surviving in the control at the end of the test was 74.5 with a variation coefficient of 32.0 %. No ephippia were observed during the test.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Formulation analysis
The test concentrations were analytically determined at the start and at the end of the renewal periods during the first week and weekly thereafter, and compared with QC samples. The geometric mean measured concentrations of test item were 0.03, 0.1, 0.3, 1 and 3 mg/L. The differences between the fresh solution concentrations of the formulations, and the concentrations measured at the end of the renewal periods, were between 90 and 132%, proving that the test item was stable in the aquatic media under the study conditions.

Observation of animals
Mortality of parent animals
No mortality occurred in the control group.
There were test item-related mortality in the test item treated groups (see Table 4).
The body size of the animals at the concentration level of 3.0 mg/L was smaller compared to the control from Day 8.

Reproduction of juveniles
The mean number of live offspring per parent animal surviving in the control at the end of the test was 74.5 with a variation coefficient of 32.0 %.
The reproduction of the surviving parent animals was significantly reduced at the concentration levels of 1 and 3 mg/L compared to the control group, where adult mortality and growth was also significantly affected.
The mean of living offspring per surviving parent animal at the end of the test (Day 21) is summarised in Table 5.
Numbers of immobilised offspring were observed during the study are summarized in Table 6.

Body length of parent animals
The body length of the surviving parent animals was significantly decreased at the concentration levels of 1 and 3 mg/L compared to the control group.
The group mean body lengths of the surviving daphnids per concentration measured at the end of the test and the relative reduction of body lengths compared to the control are summarised in Table 7.

Age of first reproduction
Statistically significant difference was observed at the concentration levels of 1 and 3 mg/L compared to the control group (see in Table 8).
In the controls, the first brood appeared on day 8, while at the highest concentration level on day 9. Therefore, it can be concluded that the test material caused a slight delay in onset of reproduction at a concentration causing significant adult mortality.

DETERMINATION OF EFFECT CONCENTRATIONS
Table 9 shows the effect parameters based on measured concentrations.

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

There was no mortality in the control group. The mean number of live offspring per parent animal surviving in the control at the end of the test was 74.5 with a variation coefficient of 32.0 %. No ephippia were observed during the test.

Table 1- Effect parameters

Endpoint	Nominal loading rate (mg/L)
	NOEC	LOEC	EC10	EC20	EC50
Reproduction	0.3	1	0.29	0.42	0.83
95% confidence intervals	-	-	0.10 – 0.89	0.14 – 1.29	0.20 – 3.25
Mortality	0.3	1	0.25	0.45	1.38
95% confidence intervals	-	-	0.04 – 0.50	0.13 – 0.84	0.73 – 3.96
Growth	0.3	1	1.19	2.09	6.14
95% confidence intervals	-	-	0.90 – 1.57	1.53 – 2.88	3.51 –10.47

 

Table 2 - Results of the Preliminary Range-Finding Test (after 14 days)

Saturated solution (SS) of nominal loading rate of 100 mg/L [%]	Untreated control	1	10	100
Number of treated / immobilised animals	5 / 0	5 / 0	5 / 0	5 / 5
Number of living offsprings	378	449	331	0
Number of living offsprings	75.6	89.8	66.2	0
[mean]
Reduction of reproduction (%)	-	-18.8	12.4	100

 

Table 3 - Definitive test: Mortality of the first definitive test (after 8 days)

Nominal loading rate [%]	Untreated control	6.25	12.5	25	50	100
Number of treated / immobilised animals	1/10	5/10	5/10	8/10	10/10	10/10

 

Table 4 - Mortality of the definitive test (after 21 days)

Nominal loading rate [%]	Untreated control	0.03	0.1	0.3	1	3
Number of treated / immobilised animals	0/10	1/10	0/10	1/10	6/10	6/10

 

Table 5 - Summary of reproductive output of the parent animals

Nominal loading rate [%]	Mean number of living offspring per surviving parent animal	SD	CV (%)	Reduction (%)	Significance
Control	74.5	23.8	32.0	-	-
0.03	84.7	21.4	25.3	-13.7	n.s.
0.1	72.4	36.5	50.4	2.8	n.s.
0.3	71.9	23.1	32.1	3.5	n.s.
1	28.3	29.6	104.9	62.1	*
3	10.5	2.1	19.8	85.9	*

SD: standard deviation
CV (%): variation coefficient
n.s.: statistically not significantly different compared to the control (Williams Multiple t-Test, α=0.05)
*: statistically significant compared to the control (but with significant adult mortality & growth affected).

Table 6 - Incidence of Immobilised Offspring

Nominal loading rate [%]	Number of Immobile offspring
Control	106
0.03	145
0.1	45
0.3	122
1	33
3	11

 

Table 7 - Mean body length of the survival parent animals at the end of the test

Nominal loading rate [%]	Mean (mm)	SD	CV (%)	Decrease (%)	Significance
Control	3.6	0.1	3.5	-	-
0.03	3.6	0.1	2.7	0.3	n.s.
0.1	3.6	0.1	1.8	-0.3	n.s.
0.3	3.6	0.2	5.1	0.1	n.s.
1	3.3	0.2	6.8	8.3	*
3	2.6	0.1	5.1	28.6	*

SD: standard deviation
CV (%): variation coefficient
n.s.: statistically not significantly different compared to the control Williams Multiple t-Test, α=0.05)
*: statistically significant compared to the control.

Table 8 - Summary of reproductive output of the parent animals

Nominal loading rate [%]	Mean number of living offspring per surviving parent animal per day	SD	CV (%)	Significance
Control	8.6	0.3	3.7	-
0.03	8.8	0.5	5.5	n.s.
0.1	9.5	1.6	17.2	n.s.
0.3	8.9	0.7	7.9	n.s.
1	10.1	1.3	13.3	*
3	10.1	0.7	7.2	*

SD: standard deviation
CV (%): variation coefficient
n.s.: statistically not significantly different compared to the control (Welsh-t-test, α=0.05)
*: statistically significant compared to the control.

Table 9 - Effect parameters

Endpoint	Nominal loading rate (mg/L)
	NOEC	LOEC	EC10	EC20	EC50
Reproduction	0.3	1	0.29	0.42	0.83
95% confidence intervals	-	-	0.10 – 0.89	0.14 – 1.29	0.20 – 3.25
Mortality	0.3	1	0.25	0.45	1.38
95% confidence intervals	-	-	0.04 – 0.50	0.13 – 0.84	0.73 – 3.96
Growth	0.3	1	1.19	2.09	6.14
95% confidence intervals	-	-	0.90 – 1.57	1.53 – 2.88	3.51 –10.47

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study, the test item caused significant mortality and reduced body length at the concentration levels of 1 and 3 mg/L. At concentrations with significant adult toxicity, the age of first reproduction and the reproductive capacity of the parental daphnids were affected. At concentrations with no evident adult toxicity (≤0.3 mg/L) there were no reproductive effects observed.

Executive summary:

The purpose of this study was to estimate the effect of the test item on the reproductive output of Daphnia magna determining the NOEC, LOEC, EC10, EC20 and EC50 for reproduction after 21 days of exposure.
Daphnia magna were exposed in a semi-static test to saturated aqueous test media containing the test item at 0.03, 0.1, 0.3, 1 and 3 mg/L nominal loading rate in a limit test for 21 days.
The test method of application and the test species Daphnia magna are recommended by the test guidelines.
Concentration of the test item in the test solution was determined - when freshly prepared and at renewal - on one occasion during the first week (at the start of the experiment) then, these determinations were repeated at weekly intervals thereafter (in total, 4 renewal periods were measured) based on the performed analytical method validation. Fully defined Elendt M4 medium was used as dilution water for the test solution. The geometric mean measured concentrations of test item were the same as the nominal concentrations; 0.03, 0.1, 0.3, 1 and 3 mg/L.
Each exposure and control group comprised 10 Daphnids. The test animals were maintained individually, one per test vessel, with ~80 mL of test solution in each vessel.
The time to the production of first brood and the numbers of broods per animal were recorded during the test. The measurement of the length of the surviving parent animals was performed at the end of the test.
There were test item-related mortality at the concentration levels of 0.03, 0.3, 1 and 3 mg/L during the experiment.
The average cumulative number of young per parent animal in the control was 74.5 with a variation coefficient of 32.0 %.
Immobilised offspring were observed during the study.
The reproduction of the surviving parent animals was significantly reduced at the concentration levels of 1 and 3 mg/L compared to the control group.
At the same concentration levels (1 and 3 mg/L) statistically significant reduced body length of the surviving parent animals was observed and the age of first reproduction was significantly different.
The effected parameters based on measured concentrations are detailed in Table 1.

In conclusion, under the conditions of the present study, Omnirad 184 Hydroxycyclohexyl phenyl ketone - EC number: 213- 426-9, caused significant mortality and reduced body length at the concentration levels of 1 and 3 mg/L. At concentrations with significant adult toxicity, the age of first reproduction and the reproductive capacity of the parental daphnids were affected. At concentrations with no evident adult toxicity (≤0.3 mg/L) there were no reproductive effects observed.

 

Description of key information

Omnirad 184 Hydroxycyclohexyl phenyl ketone - EC number: 213- 426-9, caused significant mortality and reduced body length at the concentration levels of 1 and 3 mg/L. At concentrations with significant adult toxicity, the age of first reproduction and the reproductive capacity of the parental daphnids were affected. At concentrations with no evident adult toxicity (≤0.3 mg/L) there were no reproductive effects observed.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

>= 0.25 - <= 2.09 mg/L

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.3 mg/L