dec-1-en-4-yne

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:

28-08-2018 to 31-08-2018

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: June 2016 ; signature: 2017

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All relevant concentration levels and the control were analytically verified via GC-MS at the start (0 hours), 24 hours and at the end of the exposure (72 hours). Nominally: 0 (control), 0.954%, 3.05%, 9.77%, 31.3% and 100% of the saturated solution with corresponding geometric mean measured concentrations: 0 (control), 0.0152, 0.0488, 0.155, 0.494 and 2.131 mg/L (or 15.2, 48.8, 155, 494 and 2131 µg/L)
- Sampling method: Analytical evaluation of the concentrations of the test item were carried out via GC-MS from freshly prepared media after 0 hours (with algae) and old test media after 72 hours (with algae) of exposure. Three replicates per concentration and six for the control (without test item) were prepared. Separate replicates for each measuring time were prepared. All samples for the test item analysis were taken from additionally prepared replicates with algae. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item exposures was prepared from stock solution. A saturated solution with a nominal test item concentration of 0.1 mL/L was freshly prepared with dilution water prior to the start of exposure. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 24 hours with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 2 hours for separation of undissolved test item. Thereafter, the saturated solution was removed by siphoning (from the approximate bottom of the glass flask). The saturated solution was checked for undissolved test item (formation of emulsion), which was negative. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with dilution water. 5 test item concentrations in a geometric series with a separation factor of 3.2, were prepared by diluting the stock solution with dilution water as follows: : 0.954%, 3.05%, 9.77%, 31.3% and 100% of the saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.0152, 0.0488, 0.155, 0.494 and 2.131 mg/L (or 15.2, 48.8, 155, 494 and 2131 µg/L) which were based on analysis during the definitive test period.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: potassium dichromate were prepared in a separately conducted reference test (documented in the full study report). A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2590 – 5180 lux for 24 hours per day.

ACCLIMATION
- Acclimation period: No. However, a four days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): No. Culture Medium: Nutrient medium Z according to LÜTTGE et al. (1994) Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.
Dilution water: (mg/L) - NH4Cl 15 ; MgCl2.6 H2O: 12 ; CaCl2.2 H2O: 18 ; MgSO4.7H2O: 15 ; KH2PO4: 1.6 ; FeCl3.6H2O: 0.064 ; Na2EDTA.2H2O: 0.1 ; H3BO3: 0.185 ; MnCl2.4H2O: 0.415 ; ZnCl2: 3x10-3 ; Na2MoO4.2H2O: 7x10-3 ; CoCl2.6H2O: 1.5x10-3 ; CuCl2.2H2O: 1x10-5 ; NaHCO3: 50 ; NaHCO3* : 250 ; MES monohydrate*: 2665.6 ; pH 8.1 +/- 0.2. This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.
* additional compounds were added to enable sufficient growth under conditions without headspace.
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

In accordance with the OECD TG 201 guideline.

Test temperature:

Nominal range: 21 - 24 °C, controlled at ± 2°C - measured room temperature values were min: 21.8 ; max 22.5 and mean 22.2 °C

pH:

0 hours: pH 8.1 ± 0.1 (and 8.09 in control); 72 hours: pH 7.96-9.44 (definitive test concentrations) and pH 9.43 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

- Preliminary test: 0 (control), 1.0, 10.0 and 100% saturated solution (non-GLP range finding test)
- Final test: 0 (control), 0.954%, 3.05%, 9.77%, 31.3% and 100% of the saturated solution
- Equivalent geometric mean measured concentrations (with algae): 0 (control), 0.0152, 0.0488, 0.155, 0.494 and 2.131 mg/L (or 15.2, 48.8, 155, 494 and 2131 µg/L)
- Measured concentrations of the test item were in the samples with algae in the range of 0.0168 to 2.28 mg/L at the start of exposure (0 h) and in the range of 75 to 87% of the initially test item concentrations at the end of exposure (72 h). All effect values given were based on geometric mean measured concentrations after 72 hours for samples.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type: Closed - Static. Sterile headspace flasks
- Material, size, headspace, fill volume: , volume: 59 mL, with aluminium tops with PFTE seals. Minimum headspace.
- Aeration: Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: nominal: 5 x 10^3 - 10^4 and current: 6839 cells/ml
- Control end cells density: Mean (of replicates after 72 hours) 825901 cells/ml (or ca. x120 increase in cell density)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes
- 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. OECD TG 201 medium. Additionally, compounds were added to enable sufficient growth under conditions without headspace. In accordance to OECD Guidance Document No. 23.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared according to guidelines (see 'Details on test organisms' field for more details on composition).
- Culture medium different from test medium: Yes. (see 'Details on test organisms' field)
- Intervals of water quality measurement: Start and end of the test period.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60 - 120 µE/m2/s ; within ± 15 % over incubation area (or 4440 to 8880 lux)
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found at the highest preliminary test concentration level of 100% saturated solution.
- Other: Initial cell density: Microscopic evaluation of the cells was carried out at the start and end of the exposure. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.

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 (control), 1.0, 10.0 and 100% saturated solution
- 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.:

1.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 1.02 - 1.32 mg/L ; based on GMM concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.271 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I.: 0.231 - 0.308 mg/L ; based on GMM concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.344 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.: 0.321 - 0.419 mg/L ; based on GMM concentrations

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.444 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% C.I.:< 0.0152 - 0.0751 mg/L ; based on GMM concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (geom. mean)

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): Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: None reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported.
- 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 reduction (ERC50: 0-72h) was 0.559 mg/L with a 95% confidence interval ranging from 0.538 to 0.581 mg/L with headspace and 0.811 mg/L with a 95% confidence interval ranging from 0.763 to 0.883 mg/L without headspace. The EC50 for yield inhibition (EYC50: 0-72h) was 0.320 mg/L with a 95% confidence interval ranging from 0.242 to 0.341 mg/L with headspace and 0.387 mg/L with a 95% confidence interval ranging from 0.347 to 0.428 mg/L without headspace.
The results with headspace and without headspace were within the test facility SOPs (historic values).
- Other: The sensitivity of the test system was in agreement with the historical data.

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 / LOEC was determined by calculation of statistically significant differences of growth rate and yield using: a Shapiro-Wilk's Normality test and a Levene's Equal Variance test, done firstly. Monotonicity of Concentration/Response was calculated by nonparametric trend analysis by contrasts (significance level 0.05). SD Jonckheere-Terpstra test procedure rate was done with a significance level of 0.05 for growth rate.

Table 1. Results of the Preliminary Range Finding Test (non GLP, 0 - 72 hours)

Saturated solution [%]	Growth Rate Inhibition [%]	Yield Inhibition [%]
100	100	100
10.0	10	35
1.00	2	10

 

Table 2. Percentage reduction in growth rate and inhibition of yield in the definitive test (at 72 hours)

Concentration based on Saturated Solution	Geometric mean measured test item concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Yield Inhibition
[%]	[mg/L]	No.	[d-1]		[%]	[d-1]		[%]
100	2.13	1		n.a.	100		n.a.	100
		2		n.a.	100		n.a.	100
		3		n.a.	100		n.a.	100
		Mean	(+)	n.a.	100	(+)	n.a.	100
31.3	0.494	1		1.29	19		319554	61
		2		1.29	20		316453	61
		3		1.31	18		337052	59
		Mean	(+)	1.29	19	(+)	324353	60
9.77	0.155	1		1.52	5		640829	22
		2		1.51	6		617304	25
		3		1.46	8		545207	33
		Mean	(+)	1.50	6	(+)	601113	27
3.05	0.0488	1		1.57	2		755587	8
		2		1.58	1		770685	6
		3		1.59	1		787246	4
		Mean	(-)	1.58	1	(-)	771173	6
0.954	0.152	1		1.57	2		746458	9
		2		1.56	2		737972	10
		3		1.56	3		719187	12
		Mean	(-)	1.56	2	(+)	734539	10
0%	0 (Control)	1		1.64			932493
		2		1.59			792864
		3		1.60			812819
		4		1.59			794210
		5		1.60			830200
		6		1.57			751783
		Mean		1.60			819062

Negative inhibition = increase in growth

n.a. = data not determinable

Statistically significant differences of growth rates and yield compared to control values are marked (+) and non-statistically significant are marked (-)

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-EC50 (growth rate reduction) was 1.15 (C.I. 1.02 – 1.32) mg/L based on geometric mean measured concentrations. The corresponding EC10 was 0.271 (C.I. 0.231 – 0.308) mg/L and the NOEC was 0.0488 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. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal test item concentration of 0.1 mL/L was freshly prepared with dilution water prior to the start of exposure. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 24 hours with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 2 hours for separation of undissolved test item. Thereafter, the saturated solution was removed by siphoning (from the approximate bottom of the glass flask). The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of emulsion), which was negative. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with dilution water. Glass flasks without headspace were used to reduce losses of the test item. No presence of undissolved test item during preparation and during the test was detected. Six concentrations including the saturated solution were tested in a geometrical series with a dilution factor of 3.2 : 0 (control), 0.954%, 3.05%, 9.77%, 31.3% and 100% of the saturated solution with corresponding geometric mean measured concentrations: 0 (control), 0.0152, 0.0488, 0.155, 0.494 and 2.131 mg/L, respectively. 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. Concentrations were analytically verified via GC-MS at test start and the end of exposure. At the test end the test item concentrations were between 75% to 87% of the initially measured concentrations. Therefore concentrations were calculated based on geometric mean measured concentrations. All the relevant validity criteria of the test guideline were fulfilled. The test item 72h-EC50 (growth rate reduction) was 1.15 (C.I. 1.02 – 1.32) mg/L based on geometric mean measured concentrations. The corresponding EC10 was 0.271 (C.I. 0.231 – 0.308) mg/L and the NOEC was 0.0488 mg/L.

Description of key information

ErC50 (algae; growth rate) = 1.15 (C.I. 1.02 – 1.32) mg/L based on GMM concentrations, 72-hour, freshwater, OECD TG 201, 2017

ErC10 (algae; growth rate) = 0.271 (C.I. 0.231 – 0.308) mg/L based on GMM concentrations, 72-hour, freshwater, OECD TG 201, 2017

NOEC (algae; growth rate) = 0.0488 mg/L based on GMM concentrations, 72-hour, freshwater, OECD TG 201, 2017

Key value for chemical safety assessment

EC50 for freshwater algae:

1.15 mg/L

EC10 or NOEC for freshwater algae:

0.049 mg/L

Additional information

Key study : OECD TG 202, 2017 : 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. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal test item concentration of 0.1 mL/L was freshly prepared with dilution water prior to the start of exposure. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 24 hours with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 2 hours for separation of undissolved test item. Thereafter, the saturated solution was removed by siphoning (from the approximate bottom of the glass flask). The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of emulsion), which was negative. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with dilution water. Glass flasks without headspace were used to reduce losses of the test item. No presence of undissolved test item during preparation and during the test was detected. Six concentrations including the saturated solution were tested in a geometrical series with a dilution factor of 3.2 : 0 (control), 0.954%, 3.05%, 9.77%, 31.3% and 100% of the saturated solution with corresponding geometric mean measured concentrations: 0 (control), 0.0152, 0.0488, 0.155, 0.494 and 2.131 mg/L, respectively. 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. Concentrations were analytically verified via GC-MS at test start and the end of exposure. At the test end the test item concentrations were between 75% to 87% of the initially measured concentrations. Therefore concentrations were calculated based on geometric mean measured concentrations. All the relevant validity criteria of the test guideline were fulfilled. The test item 72h-EC50 (growth rate reduction) was 1.15 (C.I. 1.02 – 1.32) mg/L based on geometric mean measured concentrations. The corresponding EC10 was 0.271 (C.I. 0.231 – 0.308) mg/L and the NOEC was 0.0488 mg/L.