3-phenylpropan-1-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:

2015-08-03 to 2015-08-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP and OECD Guideline conform study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analytically verified via HPLC-DAD after 0 hours (start of the exposure) and 72 hours (end of the exposure). Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels. The stock solution of 200 mg/L was prepared by direct addition of the test item to the dilution water and agitation.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata HINDÄK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): A three days old preculture, prepared in dilution water, was used as inoculum.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Post exposure observation period:

none

Hardness:

0.24 mmol Ca+Mg/L.

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2 °C

pH:

7.59-8.31

Dissolved oxygen:

no data

Salinity:

not applicable

Conductivity:

no data

Nominal and measured concentrations:

5 concentrations were tested in a geometrical series with a dilution factor of 2.5: 5.12 - 12.8 - 32.0 - 80.0 - 200 mg/L (nominal).
Measured concentrations: 5.14 - 12.9 - 32.4 - 79.3 - 196 mg/L. The measured concentrations of the test substance in the fresh media (0 h) were in the range of 98 to 101 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 96 to 103 % of the nominal values of all concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs
- Type: open
- Test volume: 100 mL
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL, current: 6153 cells /mL
- Control end cells density: mean 2856212 cells/mL
- No. of vessels per concentration (replicates): 3 for each concentration
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day
- Light intensity and quality: 35-70 µE /(m^2 *s)

EFFECT PARAMETERS MEASURED
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the definitive test at the test item concentration of 200 mg/L.
- Other: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
Range finding study
- Test concentrations: 0.100, 1.00, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

94.1 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.:

109 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: none
- Unusual cell shape: none
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.613 mg/L (gowth rate), 0.281 mg/L (yield)

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:
Monotonicity was done by trend analysis by contrasts (significance level 0.05).
Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01.
Levene’s test on variance homogeneity was done with a significance level 0.01.
Welshs test on significant differences was done with a significance level 0.05 for yield.
William’s test on significant differences was done with a significance level 0.05 for growth rate.
All calculations were done with ToxRat Professional (ToxRat Solutions GmbH).

Table. 1: Cell densities:

 

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
200	1	6153	7953	< LOQ	< LOQ
	2	6153	6404	2970	< LOQ
	3	6153	8069	3958	< LOQ
	Mean	6153	7475	n.a.	n.a.
80	1	6153	40037	348949	2244348
	2	6153	31652	327185	2284739
	3	6153	32135	410101	2074502
	Mean	6153	34608	362078	2201196
32	1	6153	36450	477306	2326511
	2	6153	39555	423366	2325256
	3	6153	44277	419164	2503632
	Mean	6153	40094	439945	2385133
12.8	1	6153	40505	446197	2091750
	2	6153	35471	430830	1898949
	3	6153	38074	508038	2117590
	Mean	6153	38017	461688	2036096
5.12	1	6153	37043	384104	1966310
	2	6153	48072	505310	2325695
	3	6153	33439	419415	2863708
	Mean	6153	39518	436276	2385238
Control	1	6153	48467	532531	2892182
	2	6153	54118	579226	2797099
	3	6153	47630	588132	2777342
	4	6153	45202	511582	2847024
	5	6153	43669	507192	2984757
	6	6153	43597	484957	2838870
	Mean	6153	47114	533937	2856212

 

< LOQ = below the limit of quantification (2736 cells/mL)

n.a. = data not determinable

 

Table 2: Evaluation after 72 hours:Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Nominal test item concentration [mg/L]	Replicate No.	Growth rate [d-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
200	1	n.a.	100	n.a.	100
	2	n.a.	100	n.a.	100
	3	n.a.	100	n.a.	100
	Mean	(+) n.a.	n.a.	(+) n.a.	n.a.
80	1	1.97	4	2238195	21
	2	1.97	4	2278586	20
	3	1.94	5	2068349	27
	Mean	(+)1) 1.96	4	(+) 2195043	23
32	1	1.98	3	2320358	19
	2	1.98	3	2319103	19
	3	2.00	2	2497479	12
	Mean	(+) 1) 1.99	3	(+) 2378980	17
12.8	1	1.94	5	2085597	27
	2	1.91	7	1892796	34
	3	1.95	5	2111437	26
	Mean	1.93	6	(+) 2029943	29
5.12	1	1.92	6	1960157	31
	2	1.98	3	2319542	19
	3	2.05	0	2857555	0
	Mean	(+)1) 1.98	3	(-) 2379085	17
Control	1	2.05		2886029
	2	2.04		2790946
	3	2.04		2771189
	4	2.05		2840871
	5	2.06		2978604
	6	2.05		2832717
	Mean	2.05		2850059

 

< LOQ = below the limit of quantification (2736 cells/mL)

1) Inhibition < 10% is considered not biologically significant

 

n.a. = data not determinable

 

Table 3: Measured Concentrations and Percentage of the Nominal Concentration of the test substance in Fresh Medium (0 hours) and Old Medium (72 hours)

 

Nominal test item concentration [mg/L]	0 hours fresh medium without algae		72 hours fresh medium with algae
	Measured test item concentration [mg/L]	[%]	Measured test item concentration [mg/L]	[%]
200	196	98	192	96
80	79.3	99	78.1	98
32	32.4	101	31.8	99
12.8	12.9	101	< LOQ 1) 2)
5.12	5.14	100	5.26	103
Control	< LOQ		< LOQ

 

Meas. Conc. = Measured concentration of the test item (dilution factor taken into account)

% = Percent of the nominal concentration of the test item

LOQ = Limit of Quantification (1.00 mg/L test item)

1) Percent of the nominal concentration of the test item = Limit of Quantification (1.00 mg/L test item)

The test item could not recovered in the sample 12.8 mg/L on 2015-08-06 (<SYSQL). Because of this the sample 12.8 mg/L was reanalysed on 2015-08-07 (3 replicates)

2) = not plausible, a sampling error is assumed

Table 4: Inhibition of growth rate and yield of algae (mean of 3 replicates)

Nominal test item conc. [mg/L], geom. mean	% Inhibition at 72 h (growth rate)	% Inhibition at 72 h (yield)
200	100	100
80	4	23
32	3	17
12.8	6	29
5.12	3	17

Validity criteria fulfilled:

yes

Conclusions:

In a key static growth inhibition test, the fresh algae Desmodesmus subspicatus was exposed over 72 hours to the test substance according to OECD 201 (2011) under GLP conditions. The ErC50 was determined to be 109 mg/L, the ErC10 was determined to be 94.1 mg/L and the NOEC was determined to be 80 mg/L.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 (2011). The aim of the study was the determination of NOEC, LOEC, EC10-, EC20- and EC50-values of the growth rate and the yield over a period of 72 hours.The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A stock solution of 200 mg/L was prepared by direct addition of the test item to the dilution water and agitation. Five concentrations were tested in a geometrical series with a dilution factor of 2.5 (nominal): 5.12 - 12.8 - 32.0 - 80.0 - 200 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC-DAD at the start and the end of the exposure. The measured concentrations in the fresh media (0 h) were in the range of 98 to 101 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 96 to 103 % of the nominal values of all concentrations. Therefore, all effect values given are based on the nominal test item concentrations. Potassium dichromate was tested as a reference item and an EC50 value of 0.613 mg/L was obtained for this substance. For the test substance, an EC50 (72 h) of 109 mg/L based on growth rate, an NOEC (72 h) of 80 mg/L and an EC10 of 94.1 mg/L based on growth rate were determined.

Description of key information

The 72h-ErC50 was determined to be 109 mg/L, the 72h-NOEC was determined to be 80 mg/L and the 72h-ErC10 was determined to be 94.1 mg/L in a study conducted on the fresh algae Desmodesmus subspicatus.

Key value for chemical safety assessment

EC50 for freshwater algae:

109 mg/L

EC10 or NOEC for freshwater algae:

94.1 mg/L

Additional information

In a key static growth inhibition test according to OECD 201 (2011) and GLP, the fresh algae Desmodesmus subspicatus was exposed over 72 hours to the test substance with a static system. Based on a preliminary test, five concentrations 5.12 - 12.8 - 32.0 - 80.0 - 200 mg/L in the definitive test were tested. The test concentrations were monitored via HPLC-DAD at the start and the end of the exposure. The measured concentrations of test itemthe fresh media (0 h) were in the range of 98 to 101 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 96 to 103 % of the nominal values of all concentrations.The ErC50 was determined to be 109 mg/L, the 72h-ErC10 was determined to be 94.1 mg/L and the 72h-NOEC was determined to be 80 mg/L. All effect values given are based on the nominal test item concentrations and the validity criteria were fulfilled.