1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)propane-1,3-diol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-27 to 2017-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

R,R-1-(4-tert-butyl-phenyl)-3-(4-methoxy-phenyl)-propane-1,3-diol

Molecular formula:

C20H26O3

IUPAC Name:

R,R-1-(4-tert-butyl-phenyl)-3-(4-methoxy-phenyl)-propane-1,3-diol

Constituent 2

Reference substance name:

R,S-1-(4-tert-Butyl-phenyl)-3-(4-methoxy-phenyl)-propane-1,3-diol

Molecular formula:

C20H26O3

IUPAC Name:

R,S-1-(4-tert-Butyl-phenyl)-3-(4-methoxy-phenyl)-propane-1,3-diol

Constituent 3

Reference substance name:

S,S-1-(4-tert-Butyl-phenyl)-3-(4-methoxy-phenyl)-propane-1,3-diol

Molecular formula:

C20H26O3

IUPAC Name:

S,S-1-(4-tert-Butyl-phenyl)-3-(4-methoxy-phenyl)-propane-1,3-diol

Constituent 4

Reference substance name:

S,R-1-(4-tert-Butyl-phenyl)-3-(4-methoxy-phenyl)-propane-1,3-diol

Molecular formula:

C20H26O3

IUPAC Name:

S,R-1-(4-tert-Butyl-phenyl)-3-(4-methoxy-phenyl)-propane-1,3-diol

Test material form:

solid

Details on test material:

Minimum purity 95% w/w

Specific details on test material used for the study:

Designation: Art. 132720
Synonym: Avodiol
Product name: RonaCare® Pristine Bright™
CAS-No.: 955359-35-0
Batch: EH16000620
Purity (HPLC): 95% (w/w)
Appearance: Light cream, free flowing granules
Water solubility: 45.7 mg/L (± 2.8 mg/L) (OECD 105)
Stability under
test conditions: Stable
Expiry date: June 30, 2018
Storage: +15°C to +25°C

Preparation of the Test Item:
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 further 24 hours. Then the formulation was passed through a filter membrane (pore size: 0.2 µm). The filtrate was diluted with vehicle to the different test item concentrations.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Concentrations:
1.0, 3.2, 10.0, 31.6 and 100.0 mg/L

Sampling method:
Samples of the test medium and the control medium were taken directly after preparation and after 24, 48 and 72 hours and were subjected to chemical analysis. Before analysis, the algae were removed from the medium by centrifugation at a low g-force, sufficient to settle the algae. Additionally, analysis of all five test item preparations not inoculated with algae was performed daily.

Sample storage conditions before analysis:
Samples were directly analyzed without storing

Test solutions

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: 7.70
- Total hardness: 24 mg/L as CaCO3 (calculated)

Preparation of the Test Item:
The test item was prepared freshly before the exposure. For this purpose, the calibrated flask with test medium (100 mg/L) was stirred for 24 hours.

Test organisms

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): The test system used was Pseudokirchneriella subcapitata, strain 61.81 SAG (Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany). This strain was further cultivated in the laboratories of Non-Clinical Safety.
- Method of cultivation: Preparations of 100 mL each were continuously shaken in 300 mL Erlenmeyer flasks at about 120 rpm by an orbital shaker (Universalschüttler SM25, Edmund Bühler GmbH, Hechingen, Germany). The flasks were covered with air permeable steristoppers (Heinz Herenz Medizinalbedarf GmbH, Hamburg, Germany). The flasks were continuously illuminated for the exposure period. The anticipated light intensity was between 4440 and 8880 Lux and within ± 15% from the average. The illumination was achieved by fluorescent tubes (Lumilux T5 nws FLH1 HO 80W/840, Osram GmbH, Munich, Germany) installed above the orbital shaker with the flasks.

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

-

Test conditions

Hardness:

24 mg/L as CaCO3 (calculated)

Test temperature:

23.2 - 24.3°C

pH:

7.82 - 10.53

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10.0, 31.6 and 100.0 mg/L
Measured concentrations: recoveries of all test medium samples ranged from 10.7 to 53.6%

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: 3.760.437 cells per ml
- No. of vessels per concentration (replicates): 3
- 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 light intensity measured immediately before the start of the study was 7971 Lux ± 4.4% and 8087 Lux ± 4.4% at the end of the study. The mean light intensity for the samples used for analytical determination after 24 and 48 hours were were 8017 Lux ± 2.5% and 8031 Lux ± 2.7% prior to and at the end of the exposure period, respectively. Thus, the light intensity was in the anticipated range of 4440 – 8880 Lux within ± 15% from the average.


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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.115
- Nominal test concentrations: 1.0, 3.2, 10.0, 31.6 and 100.0 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 (Gado, 2015).

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.069 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

20.475 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32.548 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8.893 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

11.036 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.68 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- 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 Council Regulation (EC) No. 761/2009. Therefore, potassium dichromate (Art. 104864) was tested as positive control (Gado, 2015).

The test item Art. 104864 (potassium dichromate) showed 72h EC values which are within the recommended range (Biomass integral EC50 0.20 – 0.75 mg/L and Growth rate EC50 of 0.60 – 1.03 mg/L).

Any other information on results incl. tables

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 comprised of one control group with six replicates and five test item groups with three replicates each. The nominal test item concentrations of 1.0, 3.2, 10.0, 31.6 and 100.0 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.

The concentrations of the test item were analytically determined by HPLC-UV. Analytical controls of the test item preparations were performed at the start of the study and then daily

Results

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

	Analytical results
	0h		24h		48h		72h
Nominal	[mg/L]	%	[mg/L]	%	[mg/L]	%	[mg/L]	%
0.0 mg/L	<LOD	-	<LOD	-	<LOD	-	<LOD	-
1.0 mg/L	0.513	51.3	0.395	39.5	0.398	39.8	0.341	34.1
3.2 mg/L	1.716	53.6	1.259	39.3	1.243	38.8	1.187	37.1
10.0 mg/L	5.148	51.5	4.141	41.4	4.063	40.6	3.772	37.7
31.6 mg/L	15.158	48.0	13.509	42.8	13.246	41.9	12.534	39.7
100.0 mg/L	45.073	45.1	11.155	11.2	23.149	23.1	32.215	32.2

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

	Analytical results
	0h		24h		48h		72h
Nominal	[mg/L]	%	[mg/L]	%	[mg/L]	%	[mg/L]	%
0.0 mg/L	<LOD	-	<LOD	-	<LOD	-	<LOD	-
1.0 mg/L	0.513	51.3	0.418	41.8	0.422	42.2	0.415	41.5
100.0 mg/L	45.073	45.1	20.720	20.7	32.583	32.6	37.609	37.6

Based on the analytical results it can be assumed that the test item was not adsorbed to the surface of the algae.

As the recovery was < 80%, the EC values were calculated based on the measured geometric mean concentrations.

For the test item, the following EC values (growth rate and yield) for Pseudokirchneriella subcapitata were determined:

Parameter (0 -72h)	Growth Rate	Yield
EC10 [mg/L] 95% confidence interval	16.069 (15.181 - 16.764)	8.893 (6.765 - 10.409)
EC20 [mg/L] 95% confidence interval	20.475 (19.939 - 21.040)	11.036 (9.106 - 13.397)
EC50 [mg/L] 95% confidence interval	32.548 (30.205 - 36.136)	16.680 (15.364 - 18.123)

Conclusion

Under the conditions of the present study, the test item showed a 72h EC50 of 32.548 mg/L for growth rate and 16.680 mg/L for yield.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the test item showed a 72h EC50 of 32.548 mg/L for growth rate and 16.680 mg/L for yield.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 201. The test item, dissolved in reconstituted water, was tested in an open semi-static system. Under the conditions of the present study, the test item showed a 72h EC50 of 32.548 mg/L for growth rate and 16.680 mg/L for yield.