2,3,4-trihydroxybenzophenone

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-12-12 to 2023-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004-04-13

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 10, 17.8, 31.6, 56.2, 100 mg/L (nominal)
Analysis of the test item concentration directly after each preparation (fresh medium) and after 24 h (old medium) was performed. One sample was given to the analytical laboratory for analysis.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item was weighed in a calibrated flask and vehicle was added. The preparation was stirred with a magnetic stirrer for 3 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 μm). The filtrate was used for the preparation of the different concentrations. The pH was not adjusted.
- Controls: reconstituted water without test item
- Chemical name of vehicle: Reconstituted water (ELENDT M4 medium) was used as vehicle.
- Concentration of vehicle in test medium: not applicable
- Evidence of undissolved material: No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Straus
- Age at study initiation: less than 24 h old
- Method of breeding: Daphnids are kept individually in 100 mL glass vessels containing approximately 60 mL reconstituted water (ELENDT M4 medium) at a water temperature of 20 ± 2 °C and a 16-hour light and 8-hour dark regime to ensure similar conditions as in the experiment. The parental daphnids are fed ad libitum with unicellular green algae Pseudokirchneriella subcapitata three times per week. Offspring are removed from the vessels at least twice per week.
- Source: originally obtained from IBACON GmbH (Roßdorf, Germany), bred in test facility
- Age of parental stock: Parental daphnids are used for reproduction until they are about 6 weeks old. Thereafter, they are replaced by neonates.
- Feeding during test: No

ACCLIMATION
- Acclimation period: not applicable, as similar conditions as in the experiment are used during breeding

Before start of the experimental phase, the young daphnids were separated from their parent animals and transferred into a test vessel containing reconstituted water. The daphnids were visually inspected for motility. If daphnids did not make any swimming movements within 15 seconds after their test vessels have been gently agitated, they were considered to be immobile and were not used for the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no

Hardness:

247 -248 mg CaCO3/L

Test temperature:

18 - 22 °C

pH:

7.06 - 7.66

Dissolved oxygen:

8.37 - 8.50 mg/L (vehicle)
The oxygen concentrations were within normal range in samples during the test.

Salinity:

not applicable

Conductivity:

580 - 582 µS/cm

Nominal and measured concentrations:

0, 10, 17.8, 31.6, 56.2, 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: test tubes
- Type: open
- Material, size, headspace, fill volume: glass, 20 mL, headspace not defined, at least 20 mL
- Volume of solution: at least 20 mL
- Aeration: no
- Renewal rate of test solution: renewal after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): vehicle is reconstituted water, thus, no additional control needed

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ELENDT M4 medium) is composed of the components (analytical grade) as detailed in “Any other information on materials and methods”. The components were dissolved in fully demineralized water (Milli-Q ® Water Purification System, Merck Millipore, Darmstadt, Germany). Stock solutions of FeSO4*7H2O and Na2EDTA*2H2O were mixed and autoclaved immediately. After preparation, the reconstituted water was aerated for 24 hours.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 h dark
- Light intensity: The mean light intensities were 665 Lux and 707 Lux prior to and at the end of the exposure period, respectively.

EFFECT PARAMETERS MEASURED: mobility (daily)

VEHICLE CONTROL PERFORMED: no, vehicle is reconstituted water, thus, no additional control needed

RANGE-FINDING STUDY
A concentration dependent immobilization was seen in a semi-static pre-test with open vessels and 48 h exposure.
nominal concentration [mg/L]: number of daphnids immobilized
0: 0/20
1.0: 0/20
10.0: 0/20
100.0: 20/20

Reference substance (positive control):

no

Remarks:

No concurrent positive control was used. To demonstrate the suitability of study conditions and to check the sensitivity of the test system, potassium dichromate is tested at regular intervals as positive control substance.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

8.52 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.43 mg/L

95% CI:

2.53 - 4.6

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Immobilisation of control: In the control group, no daphnids were immobilized.

For further details refer to "Any other information on results".

Results with reference substance (positive control):

The 24-hour EC50 must be within the range of the published data of 0.6 to 1.7 mg/L (Council Regulation (EC) No. 440/2008) and 0.6 to 2.1 mg/L (OECD Guideline No. 202).

Nominal and corresponding analytical concentrations are given in the following table:

 

Nominal test item concentration [mg/L)	Analytical results								Time Weighted Mean 3) [mg/L)
	0h		24h		24h		48h
	Fresh medium		Old medium		Fresh medium		Old medium
	Meas.1) Conc. [mg/L]	[%] 2)	Meas.1) Conc. [mg/L]	[%] 2)	Meas.1) Conc. [mg/L)	[%] 2)	Meas.1) Conc. [mg/L)	[%) 2)
0.0	<LOD	-	<LOD	-	<LOD	-	<LOD	-	-
10.0	3.334	33.3	0.127	1.3	3.152	31.5	0.133	1.3	0.968
17.8	7.548	42.4	0.511	2.9	7.907	44.4	0.573	3.2	2.742
31.6	17.069	54.0	2.572	8.1	17.301	54.8	3.066	9.7	7.943
56.2	36.019	64.1	11.037	19.6	33.820	60.2	11.694	20.8	20.976
100.0	68.882	68.9	35.291	35.3	65.254	65.3	33.396	33.4	48.880

LOD = Limit of detection (0.014 mg/L)

1) Single measurement

2) Rounded to the first decimal place

3) The formula used for calculation is given in the appendix of the study report.

 

The number of immobilized daphnids per test item concentration is shown in the following table:

 

Nominal Test ltem Concentration [mg/L]	Measured Test ltem Concentration1) [mg/L]	Number of daphnids immobilized / exposed		% of daphnids immobilized
		24 hours	48 hours	24 hours	48 hours
0.0	<LOD	0/20	0/20	0 %	0 %
10.0	0.968	0/20	2/20	0 %	10 %
17.8	2.742	0/20	4/20	0 %	20 %
31.6	7.943	7/20	19/20	35 %	95 %
56.2	20.976	20/20	20/20	100 %	100 %
100.0	48.880	20/20	20/20	100 %	100 %

1) Time weighted mean

Validity criteria fulfilled:

yes

Conclusions:

The test item showed a 48h EC50 of 3.42 mg/L for the immobilization of Daphnia magna.

Executive summary:

The objective of this study was to determine the effect of the test item on the immobilization of Daphnia magna according to OECD 202. Juvenile daphnids were exposed to different concentrations of the test item (10.0, 17 .8, 31.6, 56.2 and 100.0 mg/L) and a control in an open semi-static system. 20 daphnids, divided into four replicates, each with five animals were used in the test item groups and the control group. The daphnids were observed for immobilization after 24 and 48 hours of exposure. The test item preparation (test item groups) and the vehicle (control group) will be renewed after 24 hours. Concentrations of the test item in the test medium were analyzed via HPLC with UV-detection directly after each preparation (fresh medium) and 24 h after each preparation (old medium).

Nominal and corresponding analytical concentrations are given in the following table:

Nominal test item concentration [mg/L)	Analytical results								Time Weighted Mean 3) [mg/L)
	0h		24h		24h		48h
	Fresh medium		Old medium		Fresh medium		Old medium
	Meas.1) Conc. [mg/L]	[%] 2)	Meas.1) Conc. [mg/L]	[%] 2)	Meas.1) Conc. [mg/L)	[%] 2)	Meas.1) Conc. [mg/L)	[%) 2)
0.0	<LOD	-	<LOD	-	<LOD	-	<LOD	-	-
10.0	3.334	33.3	0.127	1.3	3.152	31.5	0.133	1.3	0.968
17.8	7.548	42.4	0.511	2.9	7.907	44.4	0.573	3.2	2.742
31.6	17.069	54.0	2.572	8.1	17.301	54.8	3.066	9.7	7.943
56.2	36.019	64.1	11.037	19.6	33.820	60.2	11.694	20.8	20.976
100.0	68.882	68.9	35.291	35.3	65.254	65.3	33.396	33.4	48.880

LOD = Limit of detection (0.014 mg/L)

1) Single measurement

2) Rounded to the first decimal place

3) The formula used for calculation is given in the appendix of the study report.

 

The test item was shown to be not stable in the medium: The recovery rates of the test item in fresh medium were between 31.5 % to 68.9 %. In the old medium the recovery rates were between 1.3 % to 35.3 %. No test item was measured in the control medium. As the test item concentrations could not be maintained >80 % of the measured initial concentrations, the EC values were calculated based on the time weighted mean concentrations.

The number of immobilized daphnids per test item concentration is shown in the following table:

Nominal Test ltem Concentration [mg/L]	Measured Test ltem Concentration1) [mg/L]	Number of daphnids immobilized / exposed		% of daphnids immobilized
		24 hours	48 hours	24 hours	48 hours
0.0	<LOD	0/20	0/20	0 %	0 %
10.0	0.968	0/20	2/20	0 %	10 %
17.8	2.742	0/20	4/20	0 %	20 %
31.6	7.943	7/20	19/20	35 %	95 %
56.2	20.976	20/20	20/20	100 %	100 %
100.0	48.880	20/20	20/20	100 %	100 %

1) Time weighted mean

Concentration dependent immobilization was observed at nominal concentrations of 10.0 to 100 mg/L. For the test item the following EC values were determined based on the time weighted mean concentrations:

Test Duration	EC50	95 % Confidence lnterval
24h	8.52 mg/L	n.d.
48h	3.42 mg/L	2.53 - 4.60 mg/L

n.d.: not determined by ToxRat Professional due to mathematical reasons

 

Under the conditions of the present study, the test item showed a 48h EC50 of 3.42 mg/L for the immobilization of Daphnia magna.

Description of key information

The test item showed a 48h EC50 of 3.42 mg/L for the immobilization of Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

3.42 mg/L

Additional information

The objective of this study was to determine the effect of the test item on the immobilization of Daphnia magna according to OECD 202. Juvenile daphnids were exposed to different concentrations of the test item (10.0, 17 .8, 31.6, 56.2 and 100.0 mg/L) and a control in an open semi-static system. 20 daphnids, divided into four replicates, each with five animals were used in the test item groups and the control group. The daphnids were observed for immobilization after 24 and 48 hours of exposure. The test item preparation (test item groups) and the vehicle (control group) will be renewed after 24 hours. Concentrations of the test item in the test medium were analyzed via HPLC with UV-detection directly after each preparation (fresh medium) and 24 h after each preparation (old medium).

Nominal and corresponding analytical concentrations are given in the following table:

Nominal test item concentration [mg/L)	Analytical results								Time Weighted Mean 3) [mg/L)
	0h		24h		24h		48h
	Fresh medium		Old medium		Fresh medium		Old medium
	Meas.1) Conc. [mg/L]	[%] 2)	Meas.1) Conc. [mg/L]	[%] 2)	Meas.1) Conc. [mg/L)	[%] 2)	Meas.1) Conc. [mg/L)	[%) 2)
0.0	<LOD	-	<LOD	-	<LOD	-	<LOD	-	-
10.0	3.334	33.3	0.127	1.3	3.152	31.5	0.133	1.3	0.968
17.8	7.548	42.4	0.511	2.9	7.907	44.4	0.573	3.2	2.742
31.6	17.069	54.0	2.572	8.1	17.301	54.8	3.066	9.7	7.943
56.2	36.019	64.1	11.037	19.6	33.820	60.2	11.694	20.8	20.976
100.0	68.882	68.9	35.291	35.3	65.254	65.3	33.396	33.4	48.880

LOD = Limit of detection (0.014 mg/L)

1) Single measurement

2) Rounded to the first decimal place

3) The formula used for calculation is given in the appendix of the study report.

 

The test item was shown to be not stable in the medium: The recovery rates of the test item in fresh medium were between 31.5 % to 68.9 %. In the old medium the recovery rates were between 1.3 % to 35.3 %. No test item was measured in the control medium. As the test item concentrations could not be maintained >80 % of the measured initial concentrations, the EC values were calculated based on the time weighted mean concentrations.

The number of immobilized daphnids per test item concentration is shown in the following table:

Nominal Test ltem Concentration [mg/L]	Measured Test ltem Concentration1) [mg/L]	Number of daphnids immobilized / exposed		% of daphnids immobilized
		24 hours	48 hours	24 hours	48 hours
0.0	<LOD	0/20	0/20	0 %	0 %
10.0	0.968	0/20	2/20	0 %	10 %
17.8	2.742	0/20	4/20	0 %	20 %
31.6	7.943	7/20	19/20	35 %	95 %
56.2	20.976	20/20	20/20	100 %	100 %
100.0	48.880	20/20	20/20	100 %	100 %

1) Time weighted mean

Concentration dependent immobilization was observed at nominal concentrations of 10.0 to 100 mg/L. For the test item the following EC values were determined based on the time weighted mean concentrations:

Test Duration	EC50	95 % Confidence lnterval
24h	8.52 mg/L	n.d.
48h	3.42 mg/L	2.53 - 4.60 mg/L

n.d.: not determined by ToxRat Professional due to mathematical reasons

 

Under the conditions of the present study, the test item showed a 48h EC50 of 3.42 mg/L for the immobilization of Daphnia magna.