1,1-dimethoxy-2-phenylethane

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:

22 Nov - 25 Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006, corrected 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

No 2016/266

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Sampling method: at test start and test end with algae; samples and the control were stabilized (factor 2) with acetonitrile containing 0.2% formic acid
- Sample storage conditions before analysis: stored at room temperature

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: preparation of stock solution (400 mg test item/L with dilution water); stock solution was stirred 15 minutes with 1100 rpm at room temperature; stock solution was diluted to achieved the required test item concentrations
- Controls: yes, test medium control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: SAG 61.81
- Source: Experimental Phycology and Culture Collection of Algae at the University of Goettingen, Germany
- Age of inoculum (at test initiation): A four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 h per day. Stock cultures are cultivated on nutrient medium Z according to Lüttge et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

22.5 °C (22.0 - 23.0 °C)

pH:

7.78 - 7.92 (0 h test treatment); 8.09 - 9.18 (72 h test treatment)
7.96 (0 h control); 9.20 (control test end 72 h)

Nominal and measured concentrations:

nominal: control; 10.24; 25.6; 64.0; 160 and 400 mg/L
measured: refer to "Any other information on results incl. tables"

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (sealed with cotton wool plugs)
- Material, size, headspace, fill volume: Sterile 250 mL-Erlenmeyer flasks; sealded with cotton wool plugs; test volume: 100 mL; headspace 150 mL
- Aeration: no
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm; positioned randomly and repositioned daily
- Initial cells density: Nominal: approx 5 x 10 E+03 - 10 E+04 cells/mL; Current: 6305 cells/mL
- Control end cells density: 1932289 cells/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: OECD medium according to the guideline OECD 201
- Culture medium different from test medium: yes; stock cultures are cultivated on nutrient medium Z according to Lüttge et al. (1994)
- Intervals of water quality measurement: The pH-values were measured at the start of exposure in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 hours/day light
- Light intensity and quality: Fluorescent tubes, Osram L 36 W/865, cool daylight; light homogeneity: within ± 15% over incubation area; mean value 5725 lux (4881 - 6291 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm
- Other: Microscopic evaluation of algae cells at test start and test end.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5

Range finding study
- Test concentrations: control; 1.0; 100 and 400 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Growth rate inhibition (%): 1.0 mg/L: 1%; 100 mg/L: 4% and 400 mg/L: 100%; results in details are summarized within the table 1 in section "Any other information on material and methods incl. tables".

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

81.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval

Remarks:

64.6 -135 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

68.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval

Remarks:

44.9 - 130 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10.24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10.24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 10.24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

75.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % confidence intervals

Remarks:

64.7 - 139 mg/L

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Other: Analytical and biological results are summarized within the tables 1-2 in the section "Any other information on results incl. tables".

Results with reference substance (positive control):

- EC50 (growth rate): 0.436 mg/L (95% confidence interval: 0.414 - 0.458 mg/L)

Reported statistics and error estimates:

ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Shapiro-Wilk’s test on normal distribution was calculated with a significance level 0.01. Levene’s test on variance homogeneity was calculated with a significance level 0.01. Monotonicity of Concentration/Response was calculated by trend analysis by contrasts (significance level 0.05). SD Jonckheere-Terpsta test procedure rate was done for growth with a significance level 0.05. Calculations were carried out using software: Excel, Microsoft Corporation; SigmaPlot, Spss Inc.; GraphPad Prism, GraphPad Software, Inc. and ToxRat Version 3.2.1, ToxRat Solutions GmbH

Table 1: Cell Densities

Nominal item concentration [mg/L]	Replicate No.	Cell density [cells/mL]
		0 hours	24 hours	48 hours	72 hours
400	1	6305	4738	<LOQ cell density	<LOQ cell density
	2	6305	5575	<LOQ cell density	<LOQ cell density
	3	6305	4315	<LOQ cell density	<LOQ cell density
	Mean	6305	4876	n.a.	n.a.
160	1	6305	9141	2995	<LOQ cell density
	2	6305	8712	<LOQ cell density	<LOQ cell density
	3	6305	9299	3021	<LOQ cell density
	Mean	6305	9051	n.a.	n.a.
64.0	1	6305	16879	124088	1151699
	2	6305	21381	180790	1623007
	3	6305	17677	134034	1296595
	Mean	6305	18646	146304	1357100
25.6	1	6305	20547	137744	1181044
	2	6305	21643	187787	1655172
	3	6305	22762	181111	1448511
	Mean	6305	21651	168881	1428242
10.24	1	6305	21928	161626	1688408
	2	6305	19464	154088	1276496
	3	6305	21666	207649	1840775
	Mean	6305	21019	174454	1601893
Control	1	6305	28020	235518	1943358
	2	6305	25229	204255	1837956
	3	6305	27755	265157	2322035
	4	6305	21542	240141	1560792
	5	6305	24815	174667	2027900
	6	6305	24107	221694	1901693
	Mean	6305	25245	223572	1932289

LOQ cell density = Limit of quantification of the cell density (2736 cells/mL)

n.a. = not applicable

Table 2: Measured Concentrations and Percentage of the Nominal Concentration of the test item in Fresh Medium (0 hours) and Old Medium (72 hours) with Algae

Sampling date	0 hours fresh medium		72 hours old medium
Start of analysis
Nominal concentration of the test item [mg/L]	Test Item
	Meas. conc. [mg/L]	[%]	Meas. conc. [mg/L]	[%]
400	365	91	364	91
160	184	115	161	100
64.0	64.3	100	53.5	84
25.6	22.4	87	21.0	82
10.24	10.7	105	9.29	91
Control	< LOQ	< LOQ

Meas. Cone. = Measured concentration of the test item (dilution factor taken into account), mean value of two injections

% = Percentage of the nominal concentration of the test item

LOQ = Limit of quantification (2.00 mg test item/L)

Validity criteria fulfilled:

yes

Conclusions:

The algae study with Pseudokirchneriella subcapitata was performed according to OECD 201 and resulted in an ErC50 (72 h) of 81.3 mg/L, an ErC10 (72 h) of 68.7 mg/L and an NOEC of 10.24 mg/L. All effect values were based on the nominal concentrations.

Description of key information

ErC50 (72 h): 81.3 mg/L (OECD 201, Pseudokirchneriella subcapitata, nominal)

ErC10 (72 h): 68.7 mg/L (OECD 201, Pseudokirchneriella subcapitata, nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

81.3 mg/L

EC10 or NOEC for freshwater algae:

68.7 mg/L

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) using the freshwater algae Pseudokirchneriella subcapitata. The definitive test was carried out with five concentrations in a geometrical series with a dilution factor of 2.5: 10.24, 25.6, 64.0, 160 and 400 mg/L including a control. The measured concentrations of the test item at test start (0 h) were between 87 and 115% of the nominal values. The measured concentrations in the old media (72 h) were in the range of 82 to 100% of the nominal values. Thus, it is justified that the effect concentrations are based on the nominal concentrations. Inhibition of growth was recorded resulting in an ErC10 (72 h) of 68.7 mg/L (95 % confidence limits: 44.9 – 130 mg/L) and an ErC50 (72 h) 81.3 mg/L (95 % confidence limits: 64.6 – 135 mg/L). Inhibition of yield was recorded resulting in an EyC50 (72 h) of 75.8 mg/L (95 % confidence limits: 64.7 – 139 mg/L) and an EyC10 (72 h) of <10.24 mg/L.