7-ethoxy-4,6-difluoro-dibenzothiophen-3-ol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 21, 2019 - Mar 04, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted on March 23, 2006; Annex 5 corrected: July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

PREPARATION OF THE TEST MATERIAL
A stock 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 1 hour. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was used for the study. The pH value was not adjusted.

Analytical monitoring:

yes

Details on sampling:

Analysis of the test item concentration at the beginning of the exposure period and then daily was performed. Before analysis, the algae were separated from the medium by centrifugation at a low g-force (4000g 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:

Vehicle:

Nutrient (mg/L)
NH4Cl 15.0
MgCl2 * 6H2O 12.0
CaCl2 * 2H2O 18.0
MgSO4 * 7H2O 15.0
KH2PO4 1.60

FeCl3 * 6H2O 0.0640
Na2EDTA * 2H2O 0.100

H3BO3 0.185
MnCl2 * 4H2O 0.415
ZnCl2 0.00300
CoCl2 * 6H2O 0.00150
CuCl2 * 2H2O 0.00001
Na2MoO4 * 2H2O 0.00700

NaHCO3 50.0

- Total hardness: 24 mg/L as CaCO3 (calculated)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Pseudokirchneriella subcapitata, strain 61.81 SAG was obtained from „Pflanzenphysiologisches Institut der Universität Göttingen“, Germany.
- Method of cultivation: The permanent algae cultures, the pre-culture and the algae cultures of the study were cultivated in an air-conditioned room (U9/2110) 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 FLH1 HO 80W/840, Osram GmbH, München, Germany) installed above the flasks served for lighting.



ACCLIMATION
- Acclimation period: 72h
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

-

Hardness:

24 mg/L as CaCO3 (calculated)

Test temperature:

22.2 to 23.3°C

pH:

7.82 to 9.05

Nominal and measured concentrations:

Nominal concentrations: 100.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer glas flasks filled with 100 mL cell suspension
- Aeration: no
- Initial cells density: 10.000 cells per ml
- Control end cells density: 2 397 576 cells per ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to OECD TG 201
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: The mean light intensities for the samples used for analytical determination after 24 and 48 hours were 7209 Lux ± 1.9% and 7130 Lux ± 2.3% prior to and at the end of the exposure period, respectively. The mean light intensities were 7327 Lux ± 2.2% and 7330 Lux ± 2.4% prior to and at the end of the exposure period, respectively.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Nominal test concentration: 100 mg/L

Reference substance (positive control):

no

Remarks:

The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Council Regulation (EC) No. 761/2009. Therefore, potassium dichromate (Art. 104864) was tested as positive control.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: > 0.001 mg/L (LOD)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: > 0.0000001448 mg/L (limit of water solubility)

Details on results:

No significant effect on growth of the algae was observed at a nominal concentration of 100 mg/L.

The 72h EC50 for growth rate and yield were >0.0032 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Commission Regulation (EU) 2016/266. Therefore, potassium dichromate was tested as positive control.

Study design:
The study comprised of one control group with six replicates and five test item groups with three replicates each. The test item concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 mg/L were dissolved in 100 mL reconstituted water. The initial algae cell density in each replicate was 10 000 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.

Results:
The test item Art. 104864 (Potassium dichromate) showed 72h EC50 of 0.62 mg/L for growth rate and 0.27 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:

Under the conditions of the present study, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae.
The 72h EC50 for growth rate and yield were >0.0032 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

Executive summary:

Objective

The objective of the present study was to determine the effect of the test item on the growth and growth rate of the green algae species Pseudokirchneriella subcapitata.

Study Design

The study design included one control group with six replicates and five test item groups (nominal 10.0, 17.8, 31.6, 56.3 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 10 000 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.

Results

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

Nominal test item concentration [mg/L]	Analytical results
	0h1)		24h		48h		72h
	Meas. Conc. [mg/L]	[%]2)	Meas. Conc. [mg/L]	[%]2)	Meas. Conc. [mg/L]	[%]2)	Meas. Conc. [mg/L]	[%]2)
0.0	<LOQ	-	<LOQ	-	<LOQ	-	<LOQ	-
10.0	0.0060	0.060	<LOQ	-	<LOQ	-	<LOQ	-
17.8	0.0107	0.060	<LOQ	-	<LOQ	-	<LOQ	-
31.6	0.0185	0.059	<LOQ	-	<LOQ	-	<LOQ	-
56.3	0.0331	0.059	<LOQ	-	<LOQ	-	<LOQ	-
100.0	0.0568	0.057	<LOQ	-	<LOQ	-	<LOQ	-

LOQ = Limit of quantification (0.0025 mg/L)

1) Measured without algae

2) Percent of nominal concentration

In test media with algae, the recovery rates of the est item at 0 hours were between 0.057% to 0.060% of the nominal concentrations. After 24 hours all concentrations were < LOQ. 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								Geometric Mean1) [mg/L]
	0h		24h		48h		72h
	Meas. Conc. [mg/L]	[%]	Meas. Conc. [mg/L]	[%]	Meas. Conc. [mg/L]	[%]	Meas. Conc. [mg/L]	[%]
10.0	0.0060	0.006	<LOQ	-	<LOQ	-	<LOQ	-	0.0015
17.8	0.0107	0.011	<LOQ	-	<LOQ	-	<LOQ	-	0.0017
31.6	0.0185	0.019	<LOQ	-	<LOQ	-	<LOQ	-	0.0018
56.3	0.0331	0.033	0.0030	0.003	<LOQ	-	<LOQ	-	0.0028
100.0	0.0568	0.057	0.0030	0.003	<LOQ	-	<LOQ	-	0.0030

LOQ = Limit of quantification (0.0025 mg/L)

In test media without algae, the recovery rates of the test item at 0 hours were between 0.006% to 0.057% of the nominal concentrations. 24 hours after start of treatment in the two highest concentrations the recovery rate was 0.003%, respectively. In all other samples the concentrations were <LOQ.

The recovery after 24 hours was slightly higher in the samples not inoculated with algae compared to the samples inoculated with algae. It can be concluded from the analytical results, that the test item sticks on the algae surface, resulting in lower recoveries in the samples with algae but the algae were exposed to these concentrations analysed in the samples without algae. Thus, the concentrations determined in the samples without algae were used for evaluation.

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 concentrations below LOQ, the half of the LOQ was used for calculation of the geometric mean.

EC values:

For the test item, the following EC, LOEC and NOEC values were determined based on the geometric mean concentrations:

Parameter (0-72h)	Growth Rate	Yield
EC50 [mg/L] 95% confidence interval	>0.0032 n.d.	>0.0032 n.d.
EC20 [mg/L] 95% confidence interval	>0.0032 n.d.	>0.0032 n.d.
EC10 [mg/L] 95% confidence interval	>0.0032 n.d.	0.0030 (0.0026 – 0.0041)
LOEC [mg/L]	0.0032	0.0032
NOEC [mg/L]	0.0029	0.0029

n.d. = could not be determined

Conclusions

Under the conditions of the present study, the test item showed a 72h EC50 of >0.0032 mg/L for the growth rate and a 72h EC50 >0.0032 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata based on the geometric mean concentrations and, thus, both EC50 values are higher than the saturated solution in this study.

Description of key information

No significant effect on growth of the algae was observed at a nominal concentration of 100 mg/L. The 72h EC50 for growth rate and yield were >0.001 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.

OECD 201: EC50 (72 h) > 100 mg/L (nominal); > 0.0032 mg/L (geometric mean)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L