4-phenylbutenone

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:

2021-09-23 - 2021-10-14 (experimental phase)

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:

OECD Guideline for the Testing of Chemicals No. 211 (Adopted October 2, 2012):
'Daphnia magna Reproduction Test'.

Deviations:

yes

Remarks:

please refer to section "additional information on materials and methods incl. tables" for further information

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test item concentrations and the control
- Sampling method: Samples in a volume of 100 mL were collected for chemical determinations at exposure initiation (fresh), on days: 1 (fresh and spent), 2 (spent), 7 (fresh), 8 (spent), 14 (fresh), 15 (spent), 20 (fresh) and 21 of exposure (spent). Fresh samples were collected from freshly prepared solutions before splitting into replicates, whereas the spent samples were poured together from all replicates of spent solutions.
- Sample storage conditions before analysis: not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item in the amount of 16.0 mg was weighed in a glass crystallizer and quantitatively transferred into a volumetric glass flask of 2 L capacity by multiple washing with Elendt M7 medium (balance OHAUS PA 214 CM/1). A volume of 1000 mL of Elendt M7 medium was added. After mixing, the resulting mixture of a loading of 16 mg/L was visually transparent with visible non-dissolved particles. Therefore, the content of the flask was sonicated for 30 minutes in an ultrasonic cleaner (SONIC-10). As a result, the stock test item concentration of 16 mg/L was transparent and homogeneous without any non-dissolved particles. The highest test item concentration used for the exposure, i.e. 3.2 mg/L was prepared by dilution of stock test item concentration with Elendt M7 medium in a volumetric ratio of 1 : 4 (400 mL of 16 mg/L filled up to 2 L of Elendt M7 medium). Lower test item concentrations were prepared by sequential dilutions with Elendt M7 medium, in a volumetric ratio of 1 : 2. Lower test item concentrations were visually homogeneous and transparent.
- Controls: The control containing Elendt M7 medium only (1250 mL) was also sonicated for 30 minutes in an ultrasonic cleaner (SONIC-10).
- Test concentration separation factor: 3,0
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, the stock and test solutions were observed to be transparent and homogeneous without any non-dissolved particles.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Waterflea Daphnia magna
- Strain/clone: Daphnia magna Straus
- Age at study initiation (mean and range, SD): < 24 h
- Stage and instar at study initiation: neonates
- Method of breeding: The test animals were cultured in glass beakers with a capacity of 150 mL (one parent per vessel) containing 100 mL of Elendt M7 medium, at room temperature with daily cycle 16 h light : 8 h darkness (electronic time programmer, KANLUX). The stock solutions for the Elendt M7 medium prepared for the culture of Daphnia magna were renewed on a regular basis. The culture was fed with a suspension of algae, mixture of two algal species Pseudokirchneriella subcapitata : Desmodesmus subspicatus (in 2:1 ratio) originating from separate cultures in the Laboratory of Aquatic Organisms Toxicology. Group B vitamins and micronutrients necessary for proper growth were supplied with the lyophilized suspension of Spirulina sp..
- Source: In-house laboratory culture
- Feeding during test
- Food type: Algae suspension containing a mixture of Pseudokirchneriella subcapitata and Desmodesmus subspicatus (2:1, volume per volume) acquired from separate standard mass laboratory cultures of these species.
- Amount: In order to provide 0.2 mg organic carbon per parent Daphnia magna per day the sufficient amount of algae mixture suspension was calculated based on chemical determinations of TOC (total organic carbon) (Shimadzu TOC-L analyzer). Once the content of TOC was analysed, the algae mixture suspension of known TOC was stored in the dark in a refrigerator and used during exposure.
- Frequency: daily

ACCLIMATION
- Acclimation period: Not necessary, since the culture was maintained in the same test water and under comparable conditions.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: The parent Daphnia magna were individually transferred to beakers with freshly prepared treatments using Pasteur pipettes.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

fresh test media (control and 3.2 mg/L): 202 - 253 mg/L as CaCO3
aged test media (control and 3.2 mg/L): 191 - 262 mg/L as CaCO3

Test temperature:

19.9 - 22.5 °C

pH:

fresh test media: 7.75 - 7.99
aged test media: 7.91 - 8.59

Dissolved oxygen:

fresh test media: 7.91 - 8.59 mg/L
aged test media: 8.0 - 9.6 mg/L

Nominal and measured concentrations:

nominal test concentrations: 3.2, 1.1, 0.36, 0.12 and 0.04 mg/L plus control
time weighted mean measured test concentrations: 2.281, 0.437, 0.129, 0.037 and 0.016 mg/L plus the control

Details on test conditions:

TEST SYSTEM
- Test vessel: test beaker
- Type (delete if not applicable): Beakers were covered with transparent plastic lids in order to minimise evaporation and to prevent accidental contamination.
- Material, size, headspace, fill volume: glass beaker of 150 mL capacity containing 80 mL of either treatment or the control
- Aeration: Treatments and the control in beakers were not aerated.
- Renewal rate of test solution (frequency/flow rate): daily renewal
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The Elendt M7 medium recommended by the OECD Guideline No. 211 (2012) was used to culture the test system and as a diluent/solvent of the test item. The medium was prepared on the basis of deionised water by adding stock solutions of reagent-grade chemicals. The stock solutions for the culture of Daphnia magna were renewed on a regular basis.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: illumination in daily cycle: 16 h light : 8 h darkness
- Light intensity: mean light intensity: 1152 - 1261 lux
- Other: The conditions throughout the exposure were stabilised in a laboratory room equipped with a heating and fluorescent light source.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The offspring produced by each parent Daphnia magna was removed, counted and recorded daily. Observations for aborted broods, dead offspring or ephippia were performed. On each day of exposure also observations for mortality of parent Daphnia magna were conducted. The immobilisation of parent Daphnia magna is defined as both the immobilisation caused by the test item and also the accidental and/or inadvertent immobilisation.
After exposure termination (on day 21) every alive parent Daphnia magna was measured (i.e. body length excluding the anal spine) by microscopic observations with accuracy of ± 0.1 mm using eyepiece micrometer disc (Microscope CX40, Olympus).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.0
- Range finding study
- Test concentrations: Nominal concentrations of 100, 10, 1.0, and 0.1 mg/L plus the control were tested in a first and nominal concentrations of 50, 25, and 12.5 mg/L plus the control in a second preliminary acute test. Nominal concentrations of 15, 1.5, 0.15, and 0.015 mg/L plus the control were tested in a preliminary reproduction test.
- Results used to determine the conditions for the definitive study: The results of the acute and chronic preliminary tests including analytical monitoring as well as the results of two additional stability tests were used to determine the most meaningful concentration range and the test conditions for the definitive test.

Reference substance (positive control):

yes

Remarks:

The sensitivity of the Daphnia magna culture is monitored on regular basis in acute toxicity tests with the reference substance potassium dichromate.

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Intrinsic rate of population growth

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Final body size (body length) of parent Daphnia magna

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Intrinsic rate of population growth

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Final body size (body length) of parent Daphnia magna

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Intrinsic rate of population growth

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Final body size (body length) of parent Daphnia magna

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 2.281 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Details on results:

- Behavioural abnormalities: In all treatments, no changes of parent Daphnia magna appearance (e.g. discoloration, deformation, trapping at the surface) in comparison with the control were observed.
- Observations on body length and weight: The mean body length of parent Daphnia magna was 103.1%, 102.9%, 101.8%, 101.8%, and 100% in the respective test item concentrations of 0.04, 0.12, 0.36, 1.1, 3.2 mg/L in comparison to the control. The decrease of final body size (body length) of parent Daphnia magna was -3.1, -2.9, -1.8, -1.8, 0% in the test item concentrations of 0.04, 0.12, 0.36, 1.1, and 3.2 mg/L in comparison to the control, respectively.
- Other biological observations: In all test item concentrations and the control no aborted broods, dead offspring or ephippia were observed.
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, the stock test item concentration of 16 mg/L was transparent and homogeneous without any non-dissolved particles. Lower test item concentrations were prepared by sequential dilutions with Elendt M7 medium and were visually homogeneous and transparent.

Results with reference substance (positive control):

not specified

Reported statistics and error estimates:

Non-linear regression analysis and analyses: Shapiro-Wilk's Test on Normal Distribution, Levene's Test on Variance Homogeneity (with Residuals), Dunnett's Multiple t-test procedure, Fisher's Exact Binomial test with Bonferroni-Correction, Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm.

VALIDITY CRITERIA
In the definitive test, the following validity criteria specified in the OECD Guideline No. No. 211 were met:
- the mortality of the parent Daphnia magna (defined as immobilisation together with accidental and/or inadvertent immobilisation) in the control was 0% at exposure termination (criterion: not more than 20%)

- the mean number of living offspring produced per parent Daphnia magna surviving at exposure termination (defined as the number of living offspring produced per parent Daphnia magna alive at exposure termination, i.e. the mean cumulative number of offspring per survivor) in the control was 150.9 (criterium: higher than 60)

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD Guideline 211 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the long-term toxicity of the registered substance towards the aquatic invertebrate Daphnia magna.
In a semi-static test with daily water renewal, neonates (< 24 h) of the freshwater invertebrate Daphnia magna were exposed to the test substance at nominal concentrations of 3.2, 1.1, 0.36, 0.12 and 0.04 mg/L and a control for a period of 21 days under defined conditions. The number of offspring produced in each beaker, as well as the observations on immobilisation were reported daily. Besides, the body size of each living parent was measured at exposure termination.
The concentrations of 4-Phenyl-3-buten-2-one were chemically analysed using the validated high performance liquid chromatographic method with DAD detection. In fresh samples at exposure initiation and at renewals (on days: 1, 7, 14 and 20 of exposure). Thus, time weighted average concentrations of 2.281, 0.437, 0.129, 0.037 and 0.016 mg test item/L were determined.
Based on the obtained biological and analytical results, the overall 21 d NOEC was determined to be ≥ 2.281 mg test item/L for the observed effect parameters immobilisation of Daphnia magna, number of offspring per introduced and per survived Daphnia magna, final body lengths and intrinsic rate of population growth.

Executive summary:

The present study was performed under GLP according to OECD TG 211. The purpose of this study was to evaluate the influence of the test item 4-Phenyl-3-buten-2-one on mortality and reproduction of Daphnia magna during an exposure period of 21 days. Therefore, young female Daphnia (< 24 h old) taken from an in-house laboratory culture of the test facility were exposed in a semi-static reproduction test to aqueous test media containing the test item at nominal concentrations of 3.2, 1.1, 0.36, 0.12, 0.04 mg/L and a control (corresponding to time-weighted mean measured test item concentrations of 2.281, 0.437, 0.129, 0.037, 0.016 mg/L plus the control, respectively), for a period of 21 days under defined conditions. Ten replicates were used for each treatment and the control. During exposure Daphnia magna were held individually in replicate glass beakers of 150 mL capacity, containing 100 mL of either the treatment or the control per replicate with daily renewals. At each renewal, the parental Daphnia magna were transferred from spent to fresh treatments and the control. The offspring produced in each beaker was counted and removed daily. The observations on immobilisation of each parent were reported daily. The body size of each living parent was measured at exposure termination.

The concentrations of 4-Phenyl-3-buten-2-one were chemically analysed using the validated high performance liquid chromatographic method with DAD detection. The obtained results confirm that the test item concentrations were prepared correctly but not stable under test conditions. However, by conducting the study with daily water renewals the exposure concentrations were maintained as constant as possible.

Based on the obtained analytical and biological results, the following endpoints were determined referring to the time-weighted mean measured concentrations of the test material:

Number of living offspring produced per parent animal which does not die accidentally or inadvertently during the test (i.e. the mean cumulative number of offspring per introduced adult):

EC10/21 d = 0.73 mg/L

EC20/21 d and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L

Number of living offspring produced per survived parent Daphnia magna at exposure termination (i.e. the mean cumulative number of offspring per survivor):

EC10/21 d = 0.70 mg/L

EC20/21 d and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L

Immobilisation of parent Daphnia magna:

EC10/21 d, EC20/21 d, and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L

Final body size (body length) of parent Daphnia magna:

EC10/21 d, EC20/21 d, and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L

Intrinsic rate of population growth:

EC10/21 d, EC20/21 d, and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L

Description of key information

OECD 211 (GLP), Daphnia magna, 21 d, semi-static, NOEC >= 2.281 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 2.281 mg/L

Additional information

The present study was performed under GLP according to OECD TG 211. The purpose of this study was to evaluate the influence of the test item 4-Phenyl-3-buten-2-one on mortality and reproduction of Daphnia magna during an exposure period of 21 days. Therefore, young female Daphnia (< 24 h old) taken from an in-house laboratory culture of the test facility were exposed in a semi-static reproduction test to aqueous test media containing the test item at nominal concentrations of 3.2, 1.1, 0.36, 0.12, 0.04 mg/L and a control (corresponding to time-weighted mean measured test item concentrations of 2.281, 0.437, 0.129, 0.037, 0.016 mg/L plus the control, respectively), for a period of 21 days under defined conditions. Ten replicates were used for each treatment and the control. During exposure Daphnia magna were held individually in replicate glass beakers of 150 mL capacity, containing 100 mL of either the treatment or the control per replicate with daily renewals. At each renewal, the parental Daphnia magna were transferred from spent to fresh treatments and the control. The offspring produced in each beaker was counted and removed daily. The observations on immobilisation of each parent were reported daily. The body size of each living parent was measured at exposure termination.

The concentrations of 4-Phenyl-3-buten-2-one were chemically analysed using the validated high performance liquid chromatographic method with DAD detection. The obtained results confirm that the test item concentrations were prepared correctly but not stable under test conditions. However, by conducting the study with daily water renewals the exposure concentrations were maintained as constant as possible.

Based on the obtained analytical and biological results, the following endpoints were determined referring to the time-weighted mean measured concentrations of the test material:

Number of living offspring produced per parent animal which does not die accidentally or inadvertently during the test (i.e. the mean cumulative number of offspring per introduced adult):

EC10/21 d = 0.73 mg/L

EC20/21 d and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L

Number of living offspring produced per survived parent Daphnia magna at exposure termination (i.e. the mean cumulative number of offspring per survivor):

EC10/21 d = 0.70 mg/L

EC20/21 d and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L

Immobilisation of parent Daphnia magna:

EC10/21 d, EC20/21 d, and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L

Final body size (body length) of parent Daphnia magna:

EC10/21 d, EC20/21 d, and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L

Intrinsic rate of population growth:

EC10/21 d, EC20/21 d, and EC50/21 d were not determined

LOEC/21 d > 2.281 mg/L

NOEC/21 d ≥ 2.281 mg/L