SUMILIZER GP

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

yes

Remarks:

Initial cell biomass slightly higher than recommended (10000 instead of 2000-5000), however, algae were in exponential growth phase. Mean CV% for section-by-section specific growth rates in solvent control was >35% (validity criterion)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: undiluted filtrate of the stock solution (only before test start), 0 (solvent control), 0 (control) and the highest test concentration
- Sampling method: Duplicate samples were taken before test start and after 72 hours
- Sample storage conditions before analysis: Samples were deep-frozen (at about -20 °C) immediately after sampling.

For the 72-hour stability samples additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An undiluted filtrate of a supersaturated dispersion with the maximum concentration of dissolved test item was used. A stock solution was prepared at the start of the test by diluting 1.0 g of the test item in 10 mL N,N-dimethylformamide (DMF) and ultrasonicating for 10 minutes. 470 µL were dosed into 4700 mL test water to obtain a loading rate of 10 mg/L. The loading rate of 10 mg/L was clearly above the water
solubility limit of the test item. After 15 minutes of sonicating, the dispersion was stirred at room temperature over 96 hours. After stirring, the dispersion was filtered through a membrane filter (0.45 µm) just before test start. The undiluted filtrate of the test item dispersion was used as the test medium with the highest test concentration. Adequate aliquots of the test medium with the highest test concentration were diluted with test water (DMF was added to the test water at a concentration of 100 µL/L) to prepare the test media with the lower test item concentrations.
- Controls: control: dilution water only, solvent control: dilution water and DMF
- Chemical name of vehicle: DMF
- Concentration of vehicle in test medium: stock solution: 100%, final test solutions: 100 µL/L, control: 0%, solvent control: 100µL/L

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 86.81 SAG
- Source: supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Germany
- Age of inoculum: 4 days
- Method of cultivation: cultivated in synthetic test water, prepared according to the test guidelines

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3

Test temperature:

23 °C

pH:

8.0 - 9.0

Nominal and measured concentrations:

Nominal loading rate: 0 (control), 0 (solvent control), 10 mg/L and dilutions 1:2.2, 1:4.6, 1:10 and 1:22
Measured concentrations: < LOD (control), < LOD (solvent control), 1.07 (10 mg/L, 0 h) and < LOD (10 mg/L, 72 h)
The analytically determined test item concentration in the organic stock solution was 99% of the nominal value.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmayer flasks
- Type (delete if not applicable): closed with glass dishes
- Material, headspace, fill volume: glass, 35 mL, 15 mL
- Initial cells density: 10000 cells/mL
- Control end cells density: control: 63250000, solvent control: 72320000
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3 , solvent control: 6
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD TG 201 medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily ( pH, temperature)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 7400 lux (mean value), 6940 - 7950 lux range)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell densities were determined by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Remarks on result:

other: No adverse effect up to its solubility limit in the test water

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.1 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Remarks on result:

other: No adverse effect up to its solubility limit in the test water

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.1 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Results with reference substance (positive control):

The latest result of the positive control test showed that the toxic performance was valid and within the historical range in the laboratory.

Table 1: Algal cells during the test period

Dilution	Initially measured concentration (µg/L)	Flsask No	Density of algal cells (cell number x 10000 mL)
			24 h		48 h		72 h
Solvent control	-	1	3.0	3.3	8.2	7.8	68.4	69.2
		2	2.9	3.0	7.2	7.3	66.9	68.8
		3	2.3	2.3	7.0	6.6	74.6	75.5
		4	2.5	2.6	6.5	6.5	51.4	52.6
		5	3.3	3.0	7.7	8.1	60.2	61.7
		6	3.1	3.2	7.8	8.0	55.3	54.4
Control	-	1	4.2	4.1	11.9	12.0	66.6	68.6
		2	3.8	3.7	11.2	11.7	83.5	83.2
		3	3.4	3.7	11.5	11.7	63.4	62.6
1:22	n.a	1	3.0	3.6	9.6	8.6	57.3	56.9
		2	3.2	3.4	8.3	7.9	66.6	62.4
		3	3.2	3.5	10.1	9.9	56.8	58.9
1:10	n.a	1	3.3	3.8	9.7	9.6.	64.3	63.4
		2	3.4	3.5	10.3	9.8	71.8	71.6
		3	3.1	2.7	7.8	7.9	65.4	65.9
1:4.6	n.a	1	2.9	2.7	7.0	6.6	71.2	72.4
		2	3.1	3.2	8.1	8.2	72.4	72.5
		3	2.9	3.1	8.7	8.9	72.9	73.8
1:2.2	n.a	1	4.2	3.7	11.2	10.9	87.7	87.9
		2	3.4	3.9	9.8	10.3	87.1	87.5
		3	3.8	3.8	11.6	12.8	71.1	72.4
Undiluted filtrate	1.1	1	3.9	4.0	10.7	11.3	73.8	73.9
		2	2.9	2.9	10.1	10.1	82.9	82.2
		3	3.4	3.2	14.4	13.9	79.7	80.8

Table 1: Validity criteria for OECD 202 (2006)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	Control (n=6) : 63.25 Solvent control   (n=12) : 71.32	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	Control (n=6) : 24.9 Solvent control   (n=12): 48.9	Control: yes Solvent control: no
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	Control   (n=6): 3.07   Solvent control  (n=12): 4.59	yes

Validity criteria fulfilled:

yes

Remarks:

See Table 2 in 'any other information on results incl.tables'

Description of key information

No adverse effect up to its solubility limit in the test water

EC50 (72h): > 10 mg/L (Scenedesmus subspicatus, OECD 201, loading rate) [ > 1.1 µg/L (meas. (initial))]

NOEC (72h): ≥ 10 mg/L(Scenedesmus subspicatus, OECD 201, loading rate) [ ≥ 1.1 µg/L (meas. (initial))]

Additional information

One GLP study assessed the influence of the test item on the growth of the green algal species Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus) in a 72-hour static test according to OECD 201.

For the preparation of the test media, an adequate amount of an organic stock solution of the test item was dosed into test water to obtain a test item dispersion with a loading rate of 10 mg/L, which was clearly above the water solubility limit of the test item. This test item dispersion was stirred over 96 hours to dissolve a maximum amount of the test item in test water. After stirring, the test item dispersion was filtered through a membrane filter (pore size 0.45 µm) just before the start of the test.

The undiluted filtrate of the stock dispersion and the dilutions 1:2.2, 1:4.6, 1:10, and 1:22 were used as test media. Additionally, a control and a solvent control (100 µL/L N,N-dimethylformamide) were tested in parallel.

 

The measured test item concentrations in the analyzed test media samples from the undiluted filtrate of the stock dispersion amounted to 1.1 µg/L at the start of the test. The concentration of the test item decreased below the limit of detection of the analytical method (0.2 µg/L) at the end of the test.

Mean algal cell densities in the test media were at all counting dates nearly identical with or even slightly higher than those in the parallel control and solvent control cultures and no concentration-effect relationship was observed. Thus, the test item had clearly no inhibitory effect on the growth of D. subspicatus up to its solubility limits.

The resulting NOEC (72 h) and EC50 (72 h) were therefore ≥ 1.1 µg/L and > 1.1 µg/L, respectively based on initially measured concentrations. The NOEC (72 h) and EC50 (72 h) were ≥10 mg/L and > 10 mg/L, respectively, based on the loading rate.