2,3,4-trihydroxybenzophenone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2023-02-06 to 2023-02-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015-12-07

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 2006-03-23, Annex 5 corrected 2011-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 1.0, 3.2, 10.0, 31.6, 100.0 mg/L (nominal)
- Sampling method: Analysis of the test item concentration at the beginning of the exposure period and then daily was performed. The samples were measured undiluted and diluted based on the results of the stability. One sample was given to the analytical laboratory for analysis. Before analysis after 24h, 48h and 72h exposure, the algae were separated from the medium by centrifugation at a low g-force (4000 g for 20 minutes), sufficient to settle the algae. Additionally, analysis of all test item concentrations not inoculated with algae was performed.

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 was used as vehicle.
- Concentration of vehicle in test medium: not applicable
- Evidence of undissolved material: No

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): obtained from “Pflanzenphysiologisches Institut der Universität Göttingen”, Germany
- Age of inoculum (at test initiation): 2-4 day old pre-culture is used as inoculum
- Method of cultivation: The permanent algae cultures, the pre-culture and the algae cultures of the study are cultivated in an air-conditioned room with a water temperature of 21.0 - 24.0 °C, controlled at ± 2 °C. For cultivation, 300 mL Erlenmeyer flasks filled with 100 mL algae suspension were covered with air permeable stoppers (Heinz Herenz Medizinalbedarf GmbH, Hamburg, Germany). The cultures were shaken continuously at about 120 rpm (Universalschüttler SM25, Edmund Bühler GmbH, Hechingen, Germany) and lit between 4440 and 8880 Lux. Fluorescent tubes (Lumilux T5 nws FLH 1 HO 80W/840, Osram GmbH, München, Germany) installed above the flasks served for lighting.

ACCLIMATION
- Acclimation period: no as same conditions were used for cultivation and study
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

0.24 mmol/L (=24 mg/L) as CaCO3

Test temperature:

20.3 - 22.2 °C

pH:

6.98 - 8.22

Dissolved oxygen:

Will be provided once final report is available.

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

0, 1.0, 3.2, 10.0, 31.6, 100.0 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: open
- Material, size, headspace, fill volume: glass, 300 mL, headspace not defined, 100 mL
- Aeration: no
- Initial cells density: 10000 cells/mL
- Control end cells density: mean (72 h): 925361 (SD: 29853.8)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): negative control: 6; per concentration without algae: 1
- No. of vessels per vehicle control (replicates): see negative control, reconstituted water was used as vehicle

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water 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 were sterilized by membrane filtration (mean pore diameter 0.2 µL). After preparation, the reconstituted water was aerated for 24 hours.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: no
- Photoperiod: not specified
- Light intensity and quality: The mean light intensities were 6394 Lux ± 3.2 % and 6428 Lux ± 1.5 % prior to and at the end of the exposure period, respectively.
The mean light intensities for the samples used for analytical determination after 24 and 48 hours were 6538 Lux ± 2.6 % and 6549 Lux ± 3.4 % prior to and at the end of the exposure period of these flasks (48 hours), respectively.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter
- Other: morphological appearance by microscope

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:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.028 mg/L

95% CI:

0.026 - 0.029

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.034 mg/L

95% CI:

0.031 - 0.036

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.026 mg/L

95% CI:

0.024 - 0.028

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.029 mg/L

95% CI:

0.026 - 0.031

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.032 mg/L

95% CI:

0.03 - 0.035

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.045 mg/L

95% CI:

0.042 - 0.049

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Growth in the control: In the control group, the biomass increased by a factor of 92.5 over 72 hours. Therefore, the validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control group (section-by-section growth rates) during 72 hours was 22.46 %. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control group after 72 hours was 0.71 %. According to the OECD test guideline, the coefficient of variation must not be higher than 7 %. Thus, the validity criterion was fulfilled.
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: The test item preparations were clear yellow/yellowish solutions which changed into an orange/brownish clear solution at higher concentrations throughout the study.
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: not specified
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Please refer to attachment under "Overall remarks" for details on growth values.

Results with reference substance (positive control):

A separate study with the positive control (Potassium dichromate) showed 72 h EC50 of 0.71 mg/L for growth rate and 0.44 mg/L for yield. The EC50 for growth rate and yield obtained in this study are in the range of ± two standard deviation of the current historical means. Therefore, this study confirms the sensitivity and reliability of the test system used in the test facility.

Validity criteria fulfilled:

yes

Conclusions:

The test item showed a 72 h EC50 of 0.045 mg/L for the growth rate and 0.032 mg/L for the yield of the unicellular green algae Pseudokirchneriella subcapitata.

Executive summary:

A study according to OECD TG 201 under GLP conditions was conducted. The objective of the study was to determine the effect of the test item on the growth and growth rate of the green algae species Pseudokirchneriella subcapitata.

The study design included one control group with six replicates and five test item groups (nominal concentrations 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L) with three replicates, each containing 100 mL reconstituted water or test medium in an open static system. The initial algae cell density in each replicate was 10000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group.

Samples of the test and control media were taken directly after preparation and then daily and the test item concentration was analyzed via HPLC with UV detection.

Nominal and corresponding analytical concentrations of samples inoculated with algae are given in the following table:

 

	Analytical results
Nominal test item concentration [mg/L]	0 h 1)		24h		48h		72h		Geometrie Mean 4) [mg/L]
	Meas. 2) Conc.	[%] 3)	Meas. 2) Conc.	[%] 3)	Meas. 2) Conc.	[%] 3)	Meas. 2) Conc.	[%] 3)
	[mg/L]		[mg/L]		[mg/L]		[mg/L]
0.0	<LOD	-	<LOD	-	<LOD	-	<LOD	-	-
1.0	<LOQ	-	<LOQ	-	<LOD	-	<LOQ	-	0.020
3.2	0.049	1.5	<LOQ	-	<LOQ	-	<LOQ	-	0.027
10.0	0.462	4.6	<LOQ	-	<LOD	-	<LOD	-	0.030
31.6	13.910	44.0	<LOQ	-	<LOD	-	<LOD	-	0.053
100.0	71.807	71.8	0.611	0.6	<LOQ	-	<LOQ	-	0.265

LOQ = Limit of quantification (0.047 mg/L)

LOD = Limit of detection (0.014 mg/L)

1) Measured without algae

2) Single measurement

3) Rounded to the first decimal place

4) According to Mean concentration = antilog ((1/2(tn-t1))Ʃ^n-1_t=1[(log(conc₁)+log(conc₁₊₁))*(t₁₊₁-t₁)])

Where: t₁= initial time < t₂ < …. t_n=final time

Conc₁= initial concentration….conc_n= final concentration

 

In test media with algae, the recovery rates of the test item at 0 hours were between 71.8 % to <LOQ. The concentrations decreased very fast over a time period of 72 hours. The samples at 0 hours were taken before treatment and, thus, were not inoculated with algae. No test item was measured in the control medium.

 

Nominal and corresponding concentrations of analytical control samples without algae are given in the following table:

 

Nominal test item concentration [mg/L]	Analytical results
	0 h		24 h		48 h		72 h
	Meas. 1) Conc. [mg/L]	[%] 2)	Meas. 1) Conc. [mg/L]	[%] 2)	Meas. 1) Conc. [mg/L]	[%] 2)	Meas. 1) Conc. [mg/L]	[%] 2)
1.0	<LOQ	-	<LOQ	-	<LOD	-	<LOD	-
3.2	0.049	1.5	<LOQ	-	<LOD	-	<LOD	-
10.0	0.462	4.6	<LOQ	-	<LOQ	-	<LOD	-
31.6	13.910	44.0	0.048	0.2	<LOD	-	<LOD	-
100.0	71.807	71.8	0.527	0.5	<LOQ	-	<LOQ	-

LOQ = Limit of quantification (0.047 mg/L)

LOD = Limit of detection (0.014 mg/L)

1) Single measurement

2) Rounded to the first decimal place

 

There was no effect of the presence of algae on the measured test item concentrations in the test media at the different time points.

As the test item concentrations could not be maintained >80 % of the measured initial concentrations, the EC values were calculated based on the calculated geometric mean concentrations. For measured concentrations <LOQ, ½ LOQ (= 0.0235 mg/L) was used for the calculation of the geometric mean concentration. For measured concentrations <LOD, the LOD (= 0.014 mg/L) was used for the calculation of the geometric mean.

 

EC values:

For the test item the following NOEC, LOEC and EC values were determined:

 

Parameter (0-72h)	Growth Rate	Yield
NOEC [mg/L]	0.027	0.027
LOEC [mg/L]	0.030	0.030
EC10 [mg/L] 95 % confidence interval	0.029 (0.026- 0.031)	0.026 (0.024- 0.028)
EC20 [mg/L] 95 % confidence interval	0.034 (0.031 - 0.036)	0.028 (0.026 - 0.029)
EC50 [mg/L] 95 % confidence interval	0.045 (0.042 - 0.049)	0.032 (0.030 - 0.035)

Under the conditions of the present study, the test item showed a 72 h EC50 of 0.045 mg/L for the growth rate and 0.032 mg/L for the yield of the unicellular green algae Pseudokirchneriella subcapitata.

Description of key information

The test item showed a 72 h EC50 of 0.045 mg/L for the growth rate and 0.032 mg/L for the yield of the unicellular green algae Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.045 mg/L

EC10 or NOEC for freshwater algae:

0.029 mg/L

Additional information

A study according to OECD TG 201 under GLP conditions was conducted. The objective of the study was to determine the effect of the test item on the growth and growth rate of the green algae species Pseudokirchneriella subcapitata.

The study design included one control group with six replicates and five test item groups (nominal concentrations 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L) with three replicates, each containing 100 mL reconstituted water or test medium in an open static system. The initial algae cell density in each replicate was 10000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group.

Samples of the test and control media were taken directly after preparation and then daily and the test item concentration was analyzed via HPLC with UV detection.

Nominal and corresponding analytical concentrations of samples inoculated with algae are given in the following table:

 

	Analytical results
Nominal test item concentration [mg/L]	0 h 1)		24h		48h		72h		Geometrie Mean 4) [mg/L]
	Meas. 2) Conc.	[%] 3)	Meas. 2) Conc.	[%] 3)	Meas. 2) Conc.	[%] 3)	Meas. 2) Conc.	[%] 3)
	[mg/L]		[mg/L]		[mg/L]		[mg/L]
0.0	<LOD	-	<LOD	-	<LOD	-	<LOD	-	-
1.0	<LOQ	-	<LOQ	-	<LOD	-	<LOQ	-	0.020
3.2	0.049	1.5	<LOQ	-	<LOQ	-	<LOQ	-	0.027
10.0	0.462	4.6	<LOQ	-	<LOD	-	<LOD	-	0.030
31.6	13.910	44.0	<LOQ	-	<LOD	-	<LOD	-	0.053
100.0	71.807	71.8	0.611	0.6	<LOQ	-	<LOQ	-	0.265

LOQ = Limit of quantification (0.047 mg/L)

LOD = Limit of detection (0.014 mg/L)

1) Measured without algae

2) Single measurement

3) Rounded to the first decimal place

4) According to Mean concentration = antilog ((1/2(tn-t1))Ʃ^n-1_t=1[(log(conc₁)+log(conc₁₊₁))*(t₁₊₁-t₁)])

Where: t₁= initial time < t₂ < …. t_n=final time

Conc₁= initial concentration….conc_n= final concentration

 

In test media with algae, the recovery rates of the test item at 0 hours were between 71.8 % to <LOQ. The concentrations decreased very fast over a time period of 72 hours. The samples at 0 hours were taken before treatment and, thus, were not inoculated with algae. No test item was measured in the control medium.

 

Nominal and corresponding concentrations of analytical control samples without algae are given in the following table:

 

Nominal test item concentration [mg/L]	Analytical results
	0 h		24 h		48 h		72 h
	Meas. 1) Conc. [mg/L]	[%] 2)	Meas. 1) Conc. [mg/L]	[%] 2)	Meas. 1) Conc. [mg/L]	[%] 2)	Meas. 1) Conc. [mg/L]	[%] 2)
1.0	<LOQ	-	<LOQ	-	<LOD	-	<LOD	-
3.2	0.049	1.5	<LOQ	-	<LOD	-	<LOD	-
10.0	0.462	4.6	<LOQ	-	<LOQ	-	<LOD	-
31.6	13.910	44.0	0.048	0.2	<LOD	-	<LOD	-
100.0	71.807	71.8	0.527	0.5	<LOQ	-	<LOQ	-

LOQ = Limit of quantification (0.047 mg/L)

LOD = Limit of detection (0.014 mg/L)

1) Single measurement

2) Rounded to the first decimal place

 

There was no effect of the presence of algae on the measured test item concentrations in the test media at the different time points.

As the test item concentrations could not be maintained >80 % of the measured initial concentrations, the EC values were calculated based on the calculated geometric mean concentrations. For measured concentrations <LOQ, ½ LOQ (= 0.0235 mg/L) was used for the calculation of the geometric mean concentration. For measured concentrations <LOD, the LOD (= 0.014 mg/L) was used for the calculation of the geometric mean.

 

EC values:

For the test item the following NOEC, LOEC and EC values were determined:

 

Parameter (0-72h)	Growth Rate	Yield
NOEC [mg/L]	0.027	0.027
LOEC [mg/L]	0.030	0.030
EC10 [mg/L] 95 % confidence interval	0.029 (0.026- 0.031)	0.026 (0.024- 0.028)
EC20 [mg/L] 95 % confidence interval	0.034 (0.031 - 0.036)	0.028 (0.026 - 0.029)
EC50 [mg/L] 95 % confidence interval	0.045 (0.042 - 0.049)	0.032 (0.030 - 0.035)

Under the conditions of the present study, the test item showed a 72 h EC50 of 0.045 mg/L for the growth rate and 0.032 mg/L for the yield of the unicellular green algae Pseudokirchneriella subcapitata.