(2E)-2-methyl-3-(4-methylphenyl)prop-2-en-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:

18-10-2011 to 03-11-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met..

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Singular samples for possible analysis were taken from all test concentrations and the control; Frequency at t=0 h, t=24 h and t=72 h; further maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
- Sampling method: 2 mL volume samples were extracted; at the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer until analysis.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days (in pre-culture under the same conditions as the test).
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days pre-culture.
- Culturing media and conditions (same as test or not): No.
Stock Culture Medium M1 ; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis) with the following composition: NaNO3 500 mg/L; K2HPO4.3H2O 52 mg/L; MgSO4.7H2O 75 mg/L; Na2CO3.10H2O 54 mg/L; C6H8O7.H2O 6 mg/L; NH4NO3 330 mg/L; CaCl2.2H2O 35 mg/L; C6H5FeO7.xH2O 6 mg/L; H3BO3 2.9 mg/L; MnCl2.4H2O 1.81 mg/L; ZnCl2 0.11 mg/L; CuSO4.5H2O 0.08 mg/L; (NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-Culture and definitive test adjusted-Medium M2 : NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 7 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/l); pH 8.1 ± 0.2
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Ca+Mg: 0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 21.8 and 22.9 °C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

pH:

0 hours: pH 8.3 ± 0.1 ; 72 hours: pH 8.0 (definitive test concentrations) and pH 8.0 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Range-finder test: 0.1, 1.0, 10 and 100 mg/L with control
Final test: 0.32, 1.0, 3.2, 10, and 32 mg/L with control.
See table 3 for nominal and measured concentrations at initial exposure and during the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 100 mL, containing 50 mL test solution ; closed airtight (capped)
- Aeration: Vessel shaken continuously.
- Initial cells density: 1 x 10^4 cells/mL.
- Control end cells density: Mean (of replicates after 72 hours) 109.89 x10^4 cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. adjusted-Medium M2.
- Detailed composition if non-standard medium was used: adjusted M2 according to OECD 201 using RO-water: NH4Cl: 15 mg/L; MgCl2.6H2O: 12 mg/L; CaCl2.2H2O: 18 mg/L; MgSO4.7H2O: 15 mg/L; KH2PO4: 1.6 mg/L; FeCl3.6H2O: 64 µg/L; Na2EDTA.2H2O: 100 µg/L; H3BO3: 185 µg/L; MnCl2.4H2O: 415 µg/L; ZnCl2: 3 µg/L; CoCl2.6H2O: 1.5 µg/L; CuCl2.2H2O: 0.01 µg/L; Na2MoO4.2H2O: 7 µg/L; NaHCO3: 7 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L); HEPES buffer 6 mmol/L; pH 8.1 ± 0.2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: Yes. Medium M1 used for stock culture medium. adjusted-Medium M2 used for pre-culture medium and test medium. See above for more information.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 68 to 75 µE/m2/s.
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer at 720nm with immersion probe (pathlength = 20mm). Algal medium used as blank.
- Other: Initial cell density: Cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Three replicates per concentration were exposed to dilutions representing 0.1, 1.0, 10 and 100 mg/L in the range-finding test. In the definitive test 5 concentrations were used at 0.32, 1.0, 3.2, 10, and 32 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:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 5.1 - 30 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 1.8 - 9.1 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.92 - 5.60 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.96 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.39 - 2.3 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Yes. At low concentrations 0.13, 0.40 and 1.4 mg/L TWA yield inhibition was negative between 24-48h and/or for 0.13 mg/L TWA 0-72h. This was low dose stimulation and was limited to < 6% and was taken into account in the data analysis and effect level calculations.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Final test: During the exposure period the measured concentrations decreased in the range finder and definitive test from nominal values. No observations were made to account for the difference.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was 1.5 mg/L with a 95% confidence interval ranging from 1.1 to 2.1 mg/L. The historical ranges for growth rate inhibition using standard ‘open’ systems range between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the present test in closed systems is within the historical range. The EC50 for yield inhibition (72h-EYC50) was 0.52 mg/L with a 95% confidence interval ranging from 0.43 to 1.1 mg/L. The historical ranges for yield inhibition using standard ‘open’ systems lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the present test for open or closed systems was within range.
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.

Growth rates were in the range of the controls at TWA concentrations 0.13 and 0.40 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to 1.4 mg/L and higher were increasingly reduced. Statistically significant reduction of growth rate was found at TWA concentrations of 1.4 mg/L and higher (Bonferroni t test, α = 0.05).
Inhibition of yield increased with increasing concentration of test item from 1.4 mg/L upwards resulting in 98% inhibition at a TWA concentration of 28 mg/L. Statistically significant inhibition of yield was found at test concentrations of 1.4 mg/L and higher (Bonferroni t test, α = 0.05)

Full details of the probit analysis are reported in the full study report.

Table 1. Mean cell densities (x10^4 cells/mL) during the range-finding test

Time (h)	Test item, Nominal concentration (mg/L)
	Control	0.10	1.0	10	100
0	1.0	1.0	1.0	1.0	1.0
72	110.9	117.7	97.0	12.0	1.1

 

Table 2. Percentage reduction of growth rate and inhibition of yield during the range-finding test

Test item, Nominal concentration (mg/L)	Mean growth rate		Yield (0 – 72 h)
	μ (0-72 h)	Reduction (%)	x10^4 cells/mL	Inhibition (%)
Control (0)	0.06533	-	109.89	-
0.10	0.06621	-1.4	116.68	-6.2
1.00	0.06349	2.8	96.02	12.6
10.00	0.03439	47.4	10.96	90.0
100.00	0.00082	98.7	0.07	99.9

Table 3. Measured concentrations versus nominal concentrations: final test

Test substance, Nominal concentration (mg/L)	Measured concentration (mg/L)					TWA (mg/L)
	t=0h	t=24h	% initial	t=72 h	% initial
0.32	0.289	0.255	88	0.0140	4.8	0.13
1.0	0.914	0.868	95	0.0298	3.3	0.40
3.2	2.79	2.69	96	0.237	8.5	1.4
10	8.58	8.49	99	3.59	42	6.5
32	29.6	30.5	103	23.5	79	28
32 # WA	30.5	30.3	99	30.4	100	30

#WA – without algae

 

Table 4. Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Test item, Nominal concentration (mg/L)	Mean growth rate		Yield (0 – 72 h)
	μ (0-72 h)	Reduction (%)	x10^4 cells/mL	Inhibition (%)
Control (0)	0.07101	-	165.54	-
0.13	0.07180	-1.10	175.08	-5.80
0.40	0.07079	0.30	162.59	1.80
1.40	0.06861	3.40	139.22	15.9
6.50	0.05343	24.8	45.88	72.3
28.0	0.01818	74.4	2.74	98.3

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-ErC50 for growth rate reduction was 12.0 (C.I. 5.1 – 30.0) mg/L based on TWA concentrations. The corresponding ErC10 was 2.30 (C.I. 0.92 – 5.60) mg/L and the NOEC was 0.40 mg/L.

Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a range finding study at 0.1, 1.0, 10.0 and 100.0 mg/L nominal concentrations, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed with a concentrations of 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via HPLC-DAD at 0 hours (test start), 24 hours and at 72 hours (test end) of the exposure. The samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. At the start of the test, the actual test concentrations were in agreement with nominal (i.e. 88 - 103% of nominal). The concentrations decreased during the test. At the end of the 72-hour test period measured concentrations had decreased significantly with the highest decrease observed in the lowest concentrations. The test concentration of 32 mg/L incubated without algae no decrease was observed, it was likely that the decrease was due to adsorption of the test item to the increasing algal biomass. The range tested based on TWA concentrations corresponded to 0.13, 0.40, 1.4, 6.5 and 28 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The EC50 for growth rate reduction (72h-ErC50) was 12.0 (C.I. 5.1 – 30.0) mg/L based on TWA concentrations. The corresponding EC10 was 2.30 (C.I. 0.92 – 5.60) mg/L and the NOEC was 0.40 mg/L.

Description of key information

EC50 (aquatic algae; growth rate) = 12 mg/L (C.I. 5.1 – 30.0) based on TWA concentrations, 72 hour, freshwater, OECD TG 201, 2012

EC10 (aquatic algae; growth rate) = 2.3 mg/L (C.I. 0.93 – 5.60) based on TWA concentrations, 72 hour, freshwater, OECD TG 201, 2012

NOEC (aquatic algae; growth rate) = 0.40 mg/L based on TWA concentrations, 72 hour, freshwater, OECD TG 201, OECD TG 201, 2012

Key value for chemical safety assessment

EC50 for freshwater algae:

12 mg/L

EC10 or NOEC for freshwater algae:

0.4 mg/L

Additional information

Key data: OECD TG 201, 2012: The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a range finding study at 0.1, 1.0, 10.0 and 100.0 mg/L nominal concentrations, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed with a concentrations of 0 (control), 0.32, 1.0, 3.2, 10.0 and 32.0 mg/L. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Environmental conditions were within the acceptable limits. The concentrations of the test item were analytically verified via HPLC-DAD at 0 hours (test start), 24 hours and at 72 hours (test end) of the exposure. The samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. At the start of the test, the actual test concentrations were in agreement with nominal (i.e. 88 - 103% of nominal). The concentrations decreased during the test. At the end of the 72-hour test period measured concentrations had decreased significantly with the highest decrease observed in the lowest concentrations. The test concentration of 32 mg/L incubated without algae no decrease was observed, it was likely that the decrease was due to adsorption of the test item to the increasing algal biomass. The range tested based on TWA concentrations corresponded to 0.13, 0.40, 1.4, 6.5 and 28 mg/L. The study met the acceptability criteria prescribed by the protocol and was considered valid. The EC50 for growth rate reduction (72h-ErC50) was 12.0 (C.I. 5.1 – 30.0) mg/L based on TWA concentrations. The corresponding EC10 was 2.30 (C.I. 0.92 – 5.60) mg/L and the NOEC was 0.40 mg/L.