2,2''-dihydroxy-4,4''-(2-hydroxy-propane-1,3-diyldioxy)dibenzophenone

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 March 2023 to 03 May 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

2016

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

424-210-0

EC Name:

-

Cas Number:

23911-85-5

Molecular formula:

C29 H24 O7

IUPAC Name:

2-benzoyl-5-[3-(4-benzoyl-3-hydroxyphenoxy)-2-hydroxypropoxy]phenol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, solvent control, 0.01 mg/L at the start and at the end of each renewal period during at least the first few renewal periods.
- Sampling method: Three samples each were taken at the start and at the end of each renewal period during the test. Samples were taken from the same solution - when freshly prepared and at renewal.

Test solutions

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 100 mg/L nominal concentration was first prepared by dissolving an appropriate amount of test item in acetone. The stock solution was prepared in two steps to obtain more accurate concentration as follows:
- 1st step: 0.025 g test item was dissolved in 25 mL acetone
- 2nd step: the solution prepared in the 1st step was 10 fold diluted with acetone obtaining the 100 mg/L stock solution. This stock solution was then 10000 fold diluted with the dilution water (Elendt M4 medium) in order to obtain the test concentration of 0.01 mg/L for each water renewal step.
- Controls: The control was prepared with only dilution water (Elendt M4 Medium). The solvent control was prepared by dissolving only acetone in Elendt M4 Medium at the same concentration as in the test item solution.
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium: 0.1 mL/L.
- Evidence of undissolved material: Observing the Tyndall effect by shining a light through the solution, no presence of any colloidal matter was observed.
- Other relevant information: Based on the results obtained during analytical method validation the test item is stable for at least 8 days in acetone (stored in a freezer). Therefore, preparation of the stock solutions (used for dilution of test concentrations) was sufficient only periodically within the 8-day stability period (the stock solutions were stored in freezer until used).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age at study initiation: Less than 24 hours
- Method of breeding: Laboratory conditions
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Age of parental stock: 7 days
- Feeding during test: yes
- Food type: Concentrated algal suspension of Raphidocelis subcapitata
- Amount: 0.1 - 0.2 mg C/Daphnia/day
- Frequency: At least three times per week

ACCLIMATION
- Acclimation period: The stock animals were bred under the same conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation to avoid stressing the parent animals prior to test was not necessary.
- Health during acclimation: Apparently healthy animals were used in this test with a known history (breeding method, pre-treatment).

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Test conditions

Hardness:

249 - 258 mg/L

Test temperature:

18.7 - 20.7 °C

pH:

7.01 - 8.11

Dissolved oxygen:

7.06 - 8.93 mg/L

Nominal and measured concentrations:

Nominal concentration: 0 mg/L (Control), 0 mg/L (solvent control) 0.01 mg/L
Measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, appropriate size for holding 50-100 mL test medium.
- Test volume: 80 mL
- Aeration: no
- Renewal rate of test solution: 24-h renewal
- No. of organisms per vessel: 1 parent animal
- No. of vessels per concentration: 10
- No. of vessels per control: 10
- No. of vessels per vehicle control: 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 Medium was prepared with deionised water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: The temperature, oxygen concentration and pH values were measured at the start and at the end of each water renewal period.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 1000 -1500 lux using cool white

EFFECT PARAMETERS MEASURED: The living offspring produced by each parent animal were removed and counted daily from the appearance of the first brood. The presence of aborted eggs or dead offspring were recorded. The mortality among the parent animal was recorded daily. The length of the parent animals was measured at the end of the test.

TEST CONCENTRATIONS
- Range finding study: Based on the results of the solubility tests a preliminary experiment was performed at 0.01 mg/L concentration (i.e. limit test concentration at saturation) in order to investigate whether the test item has any effect on the reproductive output of Daphnia magna up to at least this concentration level. A concurrent solvent control ran parallel. No effects were observed at the water solubility limit (0.01 mg/L nominal concentration).
- Test concentrations: 0.01 mg/L
- Results used to determine the conditions for the definitive study: Two solubility tests were perfomed. A solubility with the Flask Method was determined at Stability: Based on the results obtained during analytical method validation the test item is not stable for at least three days in Elendt M4 Medium, therefore 24-h water renewal periods was used in order to keep the test concentration as constant as possible during the experiment.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.005 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 2

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.005 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.005 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 4

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

> 0.005 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 5

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.005 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 6

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.005 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 7

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.005 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Observations on body length at the end of the test: 4.63 - 4.68 mm
- Mortality of control: 10 % in control and solvent control
- Other adverse effects control: Replicate 1 showed very low number of offspring production, which was considered to be due to natural causes rather than a test item effect (compared to the other replicates), therefore this replicate was excluded from data analysis.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 24-h EC50: 0.79 mg/L (95 % conf. limits: 0.65 – 0.94 mg/L)

Reported statistics and error estimates:

The statistical analysis was performed by ToxRat Professional (Version 3.3.0) software.

Number of offspring (NOEC/LOEC, ECx values): To compare the two controls, two sample comparison was performed using Two-sample Mann-Whitney U-test Procedure
(alpha = 0.05, 2-sided). No statistically significant difference was found between the two groups. To determine NOEC/LOEC values for reproduction, Two-sample Mann-Whitney U-test Procedure (alpha = 0.05, one-sided smaller) was used. Pretesting on normal distribution was performed by Shapiro-Wilk´s test and on variance homogeneity by Levene’s test. To determine the ECx values for reproduction, no statistical analysis was necessary.
Mortality (NOEC/LOEC values): For evaluation of parent mortality, no statistical analysis was necessary.
Body length of parent animals: To compare the two controls, two sample comparison was performed using STUDENT-t test (alpha = 0.05, 2-sided). No statistically significant difference was found between the two groups. Comparison of body length of parents was performed using Two-sample t-test Procedure (alpha = 0.05, one-sided smaller). Pretesting on normal distribution was performed by Shapiro-Wilk’s test and on variance homogeneity by Levene’s test.
Time to production of first brood:
To compare the two controls, two sample comparison was performed using Two-sample Mann-Whitney U-test Procedure (alpha = 0.05, 2-sided). No statistically significant difference was found between the two groups.
Comparison of time to production of first brood was performed using Two-sample t-test Procedure (alpha = 0.05, one-sided greater). Pretesting on normal distribution was performed by Shapiro-Wilk’s test and on variance homogeneity by Levene’s test.

Any other information on results incl. tables

Validity criteria:
- The mortality of parent animals (female Daphnia) did not exceed 20 % in the control groups (observed mortality was 10 % in the solvent and untreated control respectively). Furthermore, the same validity criterion (mortality ≤20%), was fulfilled for accidental and inadvertent parental mortality in the test concentration (10 % mortality was observed).
- The mean number of living offspring produced per parent animal having survived at the end of the test was 168.1 (≥60) with variation coefficient of 7.7 % (≤25 %) in the solvent control and 168.3 (≥60) with variation coefficient of 19.7 % (≤25 %) in the untreated control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this 21-day chronic toxicity study no statistically significant effect on Daphnia magna at aquatic saturation (0.0052 mg/L measured TWA) were observed. The effect concentrations based on reproduction and measured time-weighted mean concentrations were determined as follow:
21-d EC10: >0.0052 mg/L
21-d NOEC: 0.0052 mg/L
21-d LOEC: >0.0052 mg/L

Executive summary:

The purpose of this study was to evaluate the influence of the test item on the reproductive output of Daphnia magna in a semi-static test system (24-h renewal periods). The test was performed according to OECD 211, EU Method C.20 and OCSPP 850.1300. Young female Daphnia (the parent animals) aged less than 24 hours at the start of the test were exposed to aqueous test media containing the test item for 21 days at the saturation concentration of 0.01 mg/L nominal (revealed in two solubility pre-tests) in a limit test (based on range finding study), in order to demonstrate that there is no statistically significant effect at this concentration level. The test item was dissolved in acetone to obtain constant initial concentrations at each water renewal period. A control and a solvent control ran in parallel. Ten Daphnia were investigated in each test group. The living offspring produced by each parent animal were removed and counted daily from the appearance of the first brood. The mortality of parent animals was recorded daily. Body length measurements of parents were performed at the end of the test. The analysis of test concentration was performed with LC-MS/MS from the test concentration and from the controls at the start and at the end of each water renewal period. No statistically significant reduction in reproduction was observed in the treatment group (saturation concentration; 0.0052 mg/L measured TWA) compared to the controls. The mean concentrations of the test item in the treatment group were in the range of 78.7 – 135 % of the nominal concentrations in the start samples and in the range of 12.9 – 32.6 % of the nominal concentrations in the end samples, therefore the endpoints were based on mean time-weighted measured concentrations. The following endpoints, based on reproduction, were determined as follow:
21-d EC10: >0.0052 mg/L
21-d NOEC: 0.0052 mg/L
21-d LOEC: >0.0052 mg/L