Methyl α-D-mannopyranoside

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:

2021-04-29 to 2021-07-19

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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L (limit test)
- Sampling method: The limit concentration and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae. At the start of the exposure, samples were taken from one additional replicate of the limit concentration and the control. At the end of the exposure, samples were taken from pooled replicates.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Limit test: 100 mg test item/L were freshly prepared with dilution water. The dispersion was agitated until the solution was visually clear.
- Controls: Dilution water without test item
- Evidence of undissolved material: none
The limit concentration was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Origin: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)
- Inoculum: 4 days old preculture

ACCLIMATION
- not specified

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

34 mg CaCO3/L

pH:

8.06 - 9.04

Salinity:

Not applicable

Conductivity:

138 µS/cm

Nominal and measured concentrations:

Nominal: 100 mg/L (results are based on nominal concentration)
Measured: 108 - 109 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs
- Type: closed
- Test volume: 100 mL
- Type: static, no renewal during test
- Initial cells density: Nominal: approximately 5E03 - 5E04 cells/mL, actual: 6864 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD TG 201 medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultrapure water was used to prepare the dilution water (conductivity max. 0.1 μS/cm).
- Total organic carbon: < 2 mg C/L
- Acidity: 0.3 mmol/L
- Alkalinity: 0.7 mmol/L
- Conductivity: 138 µS/cm
- Total hardness: 34 mg CaCO3/L

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity: Approximately 5334 to 6867 lux

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm.

TEST CONCENTRATIONS
- Definitive test: Limit test with 10 mg test item/L
- Range finding study: yes (1.00, 10.0 and 100 mg test item/L)
- Results used to determine the conditions for the definitive study: Based on range finder test

CULTURING APPARATUS
- Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm under controlled environmental conditions

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Remarks:

ErC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Remarks:

EyC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EyC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: No abnormalities
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50 (potassium dichromate) = 0.851 mg/L (95 % confidence interval: 0.802 - 0.931 mg/L)
- EyC50 (potassium dichromate) = 0.449 mg/L (95 % confidence interval: 0.396 - 0.506 mg/L)

Table 1: Measured concentrations of the test item

Sampling date	0 hours (fresh)		72 hours (aged media)
Nominal test item concentration	Measured concentration [mg/L]	% of nominal	Measured concentration [mg/L]	% of nominal
100 mg/L	108	108	109	109
Control	< LOQ		< LOQ

Table 2: Cell densities

Nominal test item concentration [mg/L]	Replicate	Cell density [cells/mL]
		0 hours	24 hours	48 hours	72 hours
100	1	6864	22921	139114	952001
	2	6864	20756	130280	853000
	3	6864	20839	149612	975076
	4	6864	25269	149207	1080959
	5	6864	21109	128552	839596
	6	6864	22573	137387	986861
	mean	6864	22245	139025	947916
Control	1	6864	23320	131143	986456
	2	6864	22602	125305	896091
	3	6864	21163	142487	1007687
	4	6864	23659	140895	887230
	5	6864	17468	136982	790523
	6	6864	24131	171517	978944
	mean	6864	22057	141388	924489

Table 3: Results of growth rate and yield after 72 hours of exposition to the test item

Nominal test item concentration [mg/L]	Replicate	Growth rate [d^-1]	Growth rate inhibition [%]	Yield [cells/mL]	Yield inhibition [%]
100	1	1.64	-1	945137	-3
	2	1.61	2	846136	8
	3	1.65	-1	968212	-6
	4	1.69	-3	1074095	-17
	5	1.60	2	832732	9
	6	1.66	-1	979997	-7
	mean	1.64	-1	941052	-3
Control	1	1.66		979592
	2	1.62		889227
	3	1.66		1000823
	4	1.62		880366
	5	1.58		783659
	6	1.65		972080
	mean	1.63		917625

The LC50 was estimated to be above the tested concentration in the limit test (> 100 mg test item/L).

 

Validity

The study meets the validity criteria of the guideline. The biomass in the control cultures increased exponentially by a factor of at least 16 within the 72-hour test period, corresponding to a specific growth rate of 0.92 day^-1 (actual value: 135-fold, specific growth rate 1.63 day^-1).

The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (actual value: 26.0 %).

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % (actual value: 1.87 %).

Validity criteria fulfilled:

yes

Conclusions:

In an Algae Growth Inhibition Test according to OECD 201, the 72-hour EC10 and EC50 towards Pseudokirchneriella subcapitata based on growth rate was estimated to be > 100 mg/L. The 72-hour EC10 and EC50 based on yield was estimated to be > 100 mg/L. The 72-hour NOEC both based on the growth rate and yield was ≥ 100 mg/L.

Executive summary:

The growth inhibiting effect of the test item towards the green algae Pseudokirchneriella subcapitata was assessed in a study according to OECD TG 201 and GLP principles. Exponentially growing cells (6864 cells per mL at start of the test) were exposed for 72 hours to the nominal concentration of 100 mg/L test item. An untreated control ran in parallel. For determination of the test item concentration, samples were taken from the test concentration and from the control at test start and test end. Test item concentrations were determined by LC-MS/MS. Biological results and endpoints are based on the nominal concentration as the measured concentrations of the test item were within ± 20 % of the nominal value. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates of the test concentration and six replicates for the control. Sealed 250 mL glass Erlenmeyer flasks filled with test liquid were used as test vessels. The cell density was measured daily via Chlorophyll a-fluorescence.
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the single treatment level (Limit test design). As a result, the 72-hour EC10 and EC50 based on growth rate was estimated to be >100 mg/L. The 72-hour EC10 and EC50 based on yield was estimated to be >100 mg/L. The 72-hour NOEC both based on the growth rate and yield was ≥ 100 mg/L. All validity criteria of the test guideline were fulfilled. The study is thus regarded valid.

Description of key information

In an Algae Growth Inhibition Test according to OECD 201, the 72-hour EC10 and EC50 towards Pseudokirchneriella subcapitata based on growth rate was estimated to be > 100 mg/L. The 72-hour EC10 and EC50 based on yield was estimated to be > 100 mg/L. The 72-hour NOEC both based on the growth rate and yield was ≥ 100 mg/L (reference 6.1.5-1). 

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The growth inhibiting effect of the test item towards the green algae Pseudokirchneriella subcapitata was assessed in a study according to OECD TG 201 and GLP principles. Exponentially growing cells (6864 cells per mL at start of the test) were exposed for 72 hours to the nominal concentration of 100 mg/L test item. An untreated control ran in parallel. For determination of the test item concentration, samples were taken from the test concentration and from the control at test start and test end. Test item concentrations were determined by LC-MS/MS. Biological results and endpoints are based on the nominal concentration as the measured concentrations of the test item were within ± 20 % of the nominal value. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates of the test concentration and six replicates for the control. Sealed 250 mL glass Erlenmeyer flasks filled with test liquid were used as test vessels. The cell density was measured daily via Chlorophyll a-fluorescence.
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the single treatment level (Limit test design). As a result, the 72-hour EC10 and EC50 based on growth rate was estimated to be >100 mg/L. The 72-hour EC10 and EC50 based on yield was estimated to be >100 mg/L. The 72-hour NOEC both based on the growth rate and yield was ≥ 100 mg/L.