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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 30 mL samples were taken from all treatments (6.25; 12.5, 25, 50, 100 mg/L) at the beginning and the end of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For the preparation of stock concentration (1 g/L) one 2 L beaker was filled with 1 L of culture medium, stirred by magnetic stirrer (300 rpm) and 1000 mg test substance were added. The preparation was stirred for 24 h and was then filtered through a filter paper.
The amounts corresponding to the test concentrations were pipetted into a graduated flask, made up to a total volume of 1000 mL with culture medium, and then transferred to the test vessels
- Evidence of undissolved material (e.g. precipitate, surface film, etc): After filtering, solutions were clear.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: 86.81
- Source (laboratory, culture collection): "Sammlung von Algenkulturen", University of Göttingen, Germany
- Method of cultivation: A slant agar tube with Desmodesmus subspicatus was stored in the refrigator. Two 300 mL conical flasks filled with 100 mL of sterile culture medium were inoculated with algae from the slant agar. Twice a week (on Mornday and thursday} the algae stock culture was transferred: 1 mL of the stock solution was diluted in two 300 mL conical flasks with 100 mL of fresh sterile culture medium. After two weeks, these stock cultures were discarded and new cultures started as described aqbove. The culture flaks were stored in an incubator. The temperature was controlled at 25 °C. The lighting supplied by fluorescent tubes Osram w.as 0. 72 E20 photons/m2s ~ 8000 lux, measured with a spherical collector. To facilitate transfer of carbon dioxide, the algal medium was continuously stirred. To ensure that stock cultures were not contaminated they were spread on a CASO agar plate with an inoculation loop once a week. The agar plates were inoculated like the submerged algal cultures. Contaminated cultures were discarded.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24.9 -25.0 °C
pH:
7.94 - 9.21
Nominal and measured concentrations:
Nominal: 6.25, 12.5, 25, 50 and 100 mg/L
Measured: Analytical results show that recovery of test substance was very low using LC-MS analysis (< 0.01 mg/L). Thus, effects were attributed to the nominal concentration.
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL conical flasks with cotton-wool plugs
- Fill volume: 150 mL
- Initial cells density: 5 E3 cells/mL
- Control end cells density: 61.3 E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous light
-Light intensity: 8000 lux

EFFECT PARAMETERS MEASURED: The cell concentration in each flask was determined after 0, 24, 48 and 72 h.
- Determination of cell concentrations: spectrophotometer (578 nm, cubette width = 50 mm)

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: Based on non-GLP pretest results the test substance was identified as poorly soluble in culture medium. The test was performed with a test substance preparation of nominal 1 g/L culture medium as stock concentration for the following dilution series: 5.25, 12.5, 25, 50 and 100 mg/L culture medium.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
760.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
biomass
Remarks on result:
other: extrapolated
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
472.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Remarks on result:
other: extrapolated
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
145.45 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
biomass
Remarks on result:
other: extrapolated
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- 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: The 6.35, 12.5, 25 and 50 mg/L treatments showed slightly higher yields and growth rates compared to the control (4 - 18%).
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test substance was poorly soluble in culture medium. After 24h stirring the test substance preparation (1 g/L) was filtered to eliminate substance deposits on the surface of the liquid and on the bottom of the beaker. A less part of the test substance was still sticking to the magnetic stirrer rod. After filtration the stock concentration was present as a clear solution.
- other: EC50 and EC10 values were extrapolated. Growth rate EC50 could not be extrapolated.

Table 1: Inhibition of yield and growth rate after 72h (nominal concentration)

Nominal concentration [mgL]

Yield

[mean cell number x E4]

Inhibition [%]

Growth rate

Inhibition

[%]

control

61.3

0

1.687

0

6.25

68.1

-11.0

1.722

-2.1

12.5

72.8

-18.9

1.744

-3.4

25

71.1

-15.9

1.736

-2.9

50

64.0

-4.4

1.700

-0.8

100

58.2

5.1

1.670

1.0

Table 2: Analytical results of LC-MS analysis

Nominal concentration [mgL]

Measured concentrations [mg/L]

0h

72h

control

0

-

6.25

< 0.01

< 0.01

12.5

< 0.01

< 0.01

25

0.013

< 0.01

50

0.023

< 0.01

100

0.044

< 0.01

Validity criteria fulfilled:
yes
Conclusions:
In a valid study according to OECD guideline 201 and performed under GLP conditions, the EL50 and EL10 values for growth rate and biomass are determined to be above the highest test concentration of 100 mg/L as loading rate WAF. By extrapolating observed effects a 72h-EC10 for growth rate is calculated to be 472.2 mg/L (nominal).
Executive summary:

The toxicity of the test substance to the algae Desmodesmus subspicatus was investigated in a study according to OECD guideline 201 under GLP. As the substance is pratically not water soluble, it was prepared by adding 6.25, 12.5, 25, 50 and 100 mg into 1000 mL culture medium, stirring for 24h and filtering. As a result, test substance preparations were clear solutions. After 72h under static conditions, only low effects (yield and biomass) were observed in the highest concetration (yield: 5.1% and growth rate: 1% inhibition). Thus, the EL50 and EL10 value for growth rate and biomass is determined to be > 100 mg/L loading rate WAF. Extrapolating the observed effects, the growth rate EC10 of the test substance to algae is calculated to be 472.2 mg/L (nominal).

Description of key information

EL50/10 (72h) > 100 mg/L (nominal, WAF) for growth rate and biomass of Desmodesmus subspicatus (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
760.7 mg/L
EC10 or NOEC for freshwater algae:
472.2 mg/L

Additional information

One study investigating the toxicity of the test substance to freshwater algae is available. The study was performed according to GLP and OECD guideline 201 using Desmodesmus subspicatus as test organism (Wehrhahn 2010). As the substance is practically not water soluble, test solutions were prepared by adding 6.25, 12.5, 25, 50 and 100 mg into 1000 mL culture medium, stirring for 24h and filtering. As a result, test substance preparations were clear solutions. After 72h under static conditions, only low effects (yield and biomass) were observed for the highest concentration (yield: 5.1% and growth rate: 1% inhibition). Thus, the EL50 and EL10 value for growth rate and biomass is determined to be > 100 mg/L loading rate WAF. Extrapolating the observed effects, the growth rate EC10 of the test substance to algae is calculated to be 472.2 mg/L (nominal).