4-(5-Propyl-tetrahydropyran-2-yl)-phenol

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:

July 28 - Nov 20, 2021

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 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 24 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 test item concentrations. The pH was not adjusted.

Analytical monitoring:

yes

Details on sampling:

samples were taken at 0 and 72 h

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

- pH: 7.9-8.6
- Total hardness: 24 mg/L as CaCO3 (calculated)

Preparation of the Test Item:
The test medium (reconstituted water and test item) was freshly prepared.
Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 µm. The filtrate was used for the study.

The present study was performed as a static test in open vessels with the following nominal concentrations:

0.05, 0.16, 0.51, 1.63, 5.22, 16.70 and 53.44 mg/L

For the control, reconstituted water was used.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 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:

21.0 to 22.2°C

pH:

7.95 to 9.06.

Nominal and measured concentrations:

Nominal concentrations: 0.05, 0.16, 0.51, 1.63, 5.22, 16.70 and 53.44 mg/L
Measured @ 0h: < LOD, 0.019, 0.062, 0.192, 0.602, 1.964, 6.518, 20.282 mg/L
Measured @ 24h: < LOD, 0.019, 0.060, 0.191, 0.602, 1.944, 6.165, 20.208 mg/L
Measured [%]
from initial 100.0, 96.8, 99.5, 100.0, 99.0, 94.6, 99.6

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: 1 379 910 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 were 5390 Lux ± 3.8% and 5402 Lux ± 1.8% 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

Nominal concentrations: 0.05, 0.16, 0.51, 1.63, 5.22, 16.70 and 53.44 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.:

1.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

2.113 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.236 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.232 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.418 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.296 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

The recoveries of test item concentrations in all test media without algae ranged from 94.6% to 100.0% from the initially measured concentrations over the time period of 72h and, thus, the test item was stable in the test medium during the study. It can be concluded from the analytical results, that the test item sticks on the algae surface, resulting in a decrease of the recoveries in the samples with algae especially at lower concentrations.
Therefore, it is reasonable to conclude, that the test item is chemically stable in the test medium and that the algae are sufficiently exposed to the test item at concentrations of ± 20% of the initially measured concentrations during the experimental phase of the study. Therefore, the EC values were calculated based on the initially measured concentrations.

Under the conditions of the present study, the test item showed a 72h EC50 of 6.236 mg/L for the growth rate and 1.296 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata.

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 0.80 mg/L for growth rate and 0.35 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.

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

Nominal test item concentration [mg/L]	Analytical results				72h [%]^3)from initial measured concentration
	0h^1)		72h
	Measuredconcentration^2) [mg/L]	[%]^3)from nominal concentration	Measuredconcentration^2) [mg/L]	[%]^3)from nominal concentration
0.0	< LOD	-	< LOD	-	-
0.05	0.019	38.0	0.014	28.0	73.7
0.16	0.062	38.8	0.049	30.6	79.0
0.51	0.192	37.6	0.147	28.8	76.6
1.63	0.602	36.9	0.453	27.8	75.2
5.22	1.964	37.6	1.612	30.9	82.1
16.70	6.518	39.0	5.659	33.9	86.8
53.44	20.282	38.0	19.838	37.1	97.8

LOD = Limit of detection (0.003 mg/L)

¹⁾Measured without algae

²⁾Single measurement

³⁾Rounded to the first decimal place

The recoveries of concentrationswithalgae slightly decreased especially in the lower concentrations over the time period of 72h from 97.8% down to 73.7% of the initially measured concentrations. The recovery concentration in the highest concentration (53.44 mg/L) with algae remained stable over the testing period. No test item was measured in the control medium.

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

Nominal test item concentration [mg/L]	Analytical results				72h [%]^2)from initial measured concentration
	0h		72h
	Measuredconcentration^1) [mg/L]	[%]^2)from nominal concentration	Measuredconcentration^1) [mg/L]	[%]^2)from nominal concentration
0.0	< LOD	-	< LOD	-	-
0.05	0.019	38.0	0.019	38.0	100.0
0.16	0.062	38.8	0.060	37.5	96.8
0.51	0.192	37.6	0.191	37.5	99.5
1.63	0.602	36.9	0.602	36.9	100.0
5.22	1.964	37.6	1.944	37.2	99.0
16.70	6.518	39.0	6.165	36.9	94.6
53.44	20.282	38.0	20.208	37.8	99.6

LOD = Limit of detection (0.003 mg/L)

¹⁾Single measurement

²⁾Rounded to the first decimal place

The recoveries of test item concentrations in all test mediawithoutalgae ranged from 94.6% to 100.0% from the initially measured concentrations over the time period of 72h and, thus, the test item was stable in the test medium during the study. It can be concluded from the analytical results, that the test item sticks on the algae surface, resulting in a decrease of the recoveries in the samples with algae especially at lower concentrations.

Therefore, it is reasonable to conclude, that the test item is chemically stable in the test medium and that the algae are sufficiently exposed to the test item at concentrations of ± 20% of the initially measured concentrations during the experimental phase of the study. Therefore, the EC values were calculated based on the initially measured concentrations.

For the test item Art. 278727 (AP-3-OL) the following EC, LOEC and NOEC values were determined:

Parameter (0-72h)	Growth rate [mg/L]	Yield [mg/L]
EC10 95% confidence interval	1.200 (0.959 – 1.443)	0.232 (0.159 – 0.307)
EC20 95% confidence interval	2.113 (1.793 – 2.426)	0.418 (0.317 – 0.519)
EC50 95% confidence interval	6.236 (5.673 – 6.859)	1.296 (1.105 – 1.520)
LOEC	0.602	0.602
NOEC	0.192	0.192

Under the conditions of the present study, the test item showed a 72h EC₅₀of 6.236 mg/L for the growth rate and 1.296 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the test item showed a 72h EC50of 6.236 mg/L for the growth rate and 1.296 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata.

Description of key information

Under the conditions of the present study, the test item showed a 72h EC₅₀of 6.236 mg/L for the growth rate and 1.296 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

EC50 for freshwater algae:

6.236 mg/L

EC10 or NOEC for freshwater algae:

1.2 mg/L