Phenol, styrenated, epoxidized

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:

2012, Sept 10 - 2013, March 24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(1-phenylethyl)-phenyloxymethyloxirane

IUPAC Name:

(1-phenylethyl)-phenyloxymethyloxirane

Test material form:

other: yellow liquid

Details on test material:

- Name of test material (as cited in study report): Novares LR 600, (1-phenylethyl)-phenyloxymethyloxirane
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: organic
- Physical state: liquid
- Analytical purity:
- Impurities (identity and concentrations):
- Composition of test material, percentage of components: Mixture of epoxidised mono- and distyrenated phenols (ratio 2:1)
- Isomers composition:
- Stability under test conditions:
- Storage condition of test material: Room temperature (20 +/- 5°C); keep away from light and humidity
- Other:

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Nominal concentration < 5 mg/L in aqueous media: 100 mL of the solution in medium was extracted with 2 mL iso-octane, and the upper phase
was measured via GC/FID. 50-fold enrichment was achieved.
- Nominal concentration ≥ 5 mg/L but < 80 mg/L in aqueous media: 20 mL of the solution in medium was extracted with 2 mL iso-octane and the
upper phase was measured via GC/FID. Tenfold enrichment was achieved.

Test solutions

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- As the solubility of the test item in medium is limited (≤10 mg/L), water-accommodated fractions were prepared for the test. This was done by
weighing of the nominal loads, adding the corresponding amount of dilution water and shaking vigorously for 24 hrs.
The resulting solutions were filtrated through 0.45 µm filters.
- During validation of th analytical method, stability in test medium eithout alga cells after 72 h had been determined as 78 % (open system) and 77 % (closed system)
- Loss of test item in the main study might be also caused by uptake of the test item by algal cells.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga, Desmodesmus subspicatus
- Strain: CHODAT
- Source: Stock culture (origin: MBM Sciencebridge GmbH, University of Göttingen in Oct 2011)
- Age of inoculum (at test initiation):
- Method of cultivation: Subcultivation from permanent culture on solid agar at 8 °C

ACCLIMATION
- Four days (96 h) before start of the test, pre-culture medium was inoculated with an aliquot of the stock culture.
- The resulting pre-culture was growing exponentially.
- Before usage the pre-culture was checked on the absence of cell aggregates

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no data

Test conditions

Hardness:

no data

Test temperature:

23 °C - 34 °C

pH:

- Start of study: 7.6 - 7.8 in treatments, 8.0 in control (without test item)
- End of study: 8.5 - 9.1 in treatments, 9.3 in control (without test item)

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

Nominal: 0.46 / 1.00 / 2.20 / 4.60 / 10.0 mg/L (loadings)
Measured: 0.50 / 1.02 / 1.83 / 2.67 / 6.37 mg/L at beginning of exposure

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks (50 ml nominal volume), unsealed
- Type: open
- Fill volume: 45 ml +/- 5 ml
- Aeration: no data
- Type of flow-through (e.g. peristaltic or proportional diluter): no data
- Renewal rate of test solution (frequency/flow rate): no data
- Initial cells density: 2206 cells/ml in each treatment
- Control end cells density: 176848 cells/ml (standard deviation: 15150) at 72 h
- No. of vessels per concentration (replicates): 3
- No. of vessels per positive control (replicates): 6
- No. of vessels per medium control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: no data
- Light intensity and quality: 5800 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Spectrophotometer; measurement of absorbtion (optical density) at 440 nm every 24 h
- Cell density was calculated based on the correlation curve between absorbtion and microscopic counts.
- Chlorophyll measurement: no data

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2 (for loadings)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 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:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.9 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:

EC50

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: AUC

Remarks on result:

other: AUC: Area Under Curve = integral of the biomass (according to EU Method C.3)

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: AUC

Remarks on result:

other: AUC: Area Under Curve = integral of the biomass (according to EU Method C.3)

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: AUC

Remarks on result:

other: AUC: Area Under Curve = integral of the biomass (according to EU Method C.3)

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: AUC

Remarks on result:

other: AUC: Area Under Curve = integral of the biomass (according to EU Method C.3)

Effect concentrations 9

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Yield: Biomass at the end minus biomass at the start of exposure (according to OECD Guideline 201)

Effect concentrations 10

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Yield: Biomass at the end minus biomass at the start of exposure (according to OECD Guideline 201)

Effect concentrations 11

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Yield: Biomass at the end minus biomass at the start of exposure (according to OECD Guideline 201)

Effect concentrations 12

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Yield: Biomass at the end minus biomass at the start of exposure (according to OECD Guideline 201)

Details on results:

- Exponential growth in the control (for algal test): yes
- No inhibition was observed up to 2.2 mg test item per L (nominal; 1.3 mg/L measured)

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The EC50 values of potassium dichromate (positive control) tested in a seperate reference test (LAUS study no. 2012 06R301) lay within the
normal range of the laboratory.
- The data showed a statistical sufficient dose-response- relation

Reported statistics and error estimates:

The measured concentrations at the start of the test tended to decrease in a dose related manner (range between 58 % and 109 %).
Therefore, the determination of biological results was based on the geometric mean of the measured concentrations.
For the estimation of the EC50 a sufficient statistical dose-response-relation was found.

Any other information on results incl. tables

Due to the limited solubility of the test item the correlation between nominal and measured concentrations - especially at the higher concentrations - was weak (see table below). This was observed in both analysed peaks.

Following tables are excerpts from the original report:

Table 10.1-d: Measured concentrations (in mg/L) and corresponding recovery rates (in %)

Nominal conc.	Measured conc. 0 h	% Nominal conc.	Measured conc. 72 h	% Nominal conc.	% Recovery	Geometric Mean
mg/l	mg/L	%	mg/ml	%	%	mg/ml
Control	< LOQ	-	< LOQ	-	-	-
0.46	0.50	108,70	0.32	69.57	64.00	0.40
1	2.02	101.5	0.59	58.50	57.64	0.77
2.2	1.83	82.95	0.97	43.86	52.88	1.33
4.6	2.67	58.04	2.26	49.13	84.64	2.46
10	6.37	63.65	5.18	51.80	81.38	5.74

Table 10.1-a: Cell Number/mL (Main Study)

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
0	Mean	2206	7905	26104	176848
0	SD	0	450	4662	15150
0.46	Mean	2206	6618	26840	169127
0.46	SD	0	1103	3874	36086
1	Mean	2206	6986	27575	161773
1	SD	0	637	2206	10008
2.2	Mean	2206	8456	31987	165818
2.2	SD	0	637	1103	13343
4.6	Mean	2206	7721	23531	75739
4.6	SD	0	1103	4592	5213
10	Mean	2206	5883	7353	8089
10	SD	0	637	3184	2776

SD = Standard Deviation

Table 10.1-e:  Growth Rate µ, Area under the Curve AUC, Yield Main Study (0 – 72 h)

Nom. Conc. in mg/L	Meas. Conc. in mg/L (geom. mean)		Growth Rate [day^-1]	AUC [Cell Conc./mL*day]	Yield [Cell Conc./mL]
0		Mean	1.46	116918	174642
		SD	0.03	11982	15150
0.46	0.40	Mean	1.44	112506	166921
		SD	0.07	15898	36086
1	0.77	Mean	1.43	109932	159567
		SD	0.02	6851	10008
2.2	1.33	Mean	1.44	117837	163612
		SD	0.03	6392	13343
4.6	2.46	Mean	1.18	63606	73533
		SD	0.02	3324	5213
10	5.74	Mean	0.42	11765	5883
		SD	0.12	4284	2776

Applicant's summary and conclusion

Validity criteria fulfilled:

yes