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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
Study period:
27 Jun - 22 Jul 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted Mar 2006, corrected 28 Jul 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 23 (Testing of Difficult Substances & Mixtures)
Version / remarks:
15 Dec 2000
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany (24 Sep 2013)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A supersaturated stock solution of 100 mg test item/L was prepared by dissolving 100.8 mg test item into 1008 mL test water. The stock solution was ultrasonicated for 15 min and shaken in an overhead shaker for 24 h. The non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). The test media were prepared just before introduction of the algae (= start of the test).
- Controls: In the control, test water (OECD medium) was used without addition of the test item.
- Evidence of undissolved material: There were no remarkable observations on the appearance of the test medium throughout the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular, freshwater green microalgae
- Strain: 61.81 SAG
- Source: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: The algae were cultivated in the laboratories of the Test Facility under standardised conditions according to the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.24 mmol/L = 24 mg/L CaCO3 (calculated value for OECD medium)
Test temperature:
0 h: 21.3 °C
72 h: 22.5 °C
pH:
0 h: 8.2 (control), 9.8 (treatment)
72 h: 7.8 (control), 9.8 (treatment)
Nominal and measured concentrations:
Control and 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: The flasks were covered with glass dishes.
- Material, size, headspace, fill volume: Fill volume: 50 mL
- Initial cells density: 5000 cells/mL
- Control end cells density: 1.20895E+06 cells/mL (72 h)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, reconstituted water (OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: The temperature was measured in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The pH was measured in all test item concentrations and the control at the start and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the OECD medium was adjusted to pH 8.1 using 1 M HCl.
- Photoperiod: continuous illumination
- Light intensity and quality: mean light intensity = 4947 lux (4500 - 5410 lux)
- Other: The test flasks were incubated in a water bath, were placed in a random order and were repositioned each day to minimize differences in test conditions.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: spectrophotometer (after 24, 48, and 72 h)
- Other: Microsopic examination of cell aspect after 72 h

TEST CONCENTRATIONS
- Range finding study: Yes, but non-GLP.
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: Effect concentration refers to max. calculated water solubility (EPI Suite)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Effect concentration refers to max. calculated water solubility (EPI Suite)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Effect concentration refers to max. calculated water solubility (EPI Suite)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: Yes, there was a 241.8-fold increase of cell density in control cultures within 72 h, which fulfills the validity criterion (≥ factor 16 within 72 h).
- Observation of abnormalities: The microscopic examination of the shape of the algal cells after 72 h did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected at the only concentration tested.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: There were no remarkable observations on the appearance of the test medium.
Results with reference substance (positive control):
- Results with reference substance valid? Yes, most recent test dating Feb 2016.
- ErC50 (72 h): 1.5 mg potassium dichromate/L (95% confidence interval: 1.43 - 1.58 mg test item/L)
Reported statistics and error estimates:
Based on the calculated cell densitites the 72 h EC50 values could not be calculated by the Probit analysis due to the lack of effects. Therefore, they were directly determined from the raw data.
For the determination of the LOEC (72 h) and NOEC (72 h), the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by a two sample t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

The influence of the test substance on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static limit test. The ErC50 and the EyC50 (72 h) were calculated to be > 4.457E-07 (nominal > 100 mg test item/L). The NOEyC (72 h), NOErC (72 h) and the associated LOEyC (72 h), LOErC (72 h) were > 4.457 E-07 mg test item/L.

All values refer to the maximim water solubility calculated with EPI Suite.

Validity criteria fulfilled:
yes
Conclusions:
No adverse effects on algae were observed up to the limit of water solubility. Thus, the 72-h EC50 and 72-h EC10 (based on growth rate) was determined to be greater than 100 mg/L (nominal). 
Executive summary:

The purpose of this test was to determine the inhibitory effect of the test item on  growth of the freshwater green algae Pseudokirchneriella subcapitata according to OECD 201. 

Exponentially growing cultures of this unicellular green algal species were exposed to the only test item concentration of nominal 100 mg/L (under defined conditions). The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.

Due to the low water solubility limit of the test item a filtrate of a supersaturated stock suspension of nominal 100 mg/L was tested. Thus, no concentration above the solubility limit of the test item in the used test water was tested.

No adverse effects on algae were observed up to the limit of water solubility. Thus, the 72-h EC50 and 72-h EC10 (based on growth rate) was determined to be greater than 100 mg/L (nominal). 

Description of key information

No adverse effects on algae were observed up to the limit of water solubility. Thus, the 72-h EC50 and 72-h EC10 (based on growth rate) was determined to be greater than 100 mg/L (nominal). 

Key value for chemical safety assessment

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