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Ecotoxicological information

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:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2009)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling schedule: Concentraion of 0.1, 1.0 and 100 mg/L were measured at 0 and 72 hours; concentration of 0 mg/L at 72 hours only.
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.
Vehicle:
no
Details on test solutions:
A stock solution was prepared to give the desired series of test concentrations. 283.7 mg (active ingredient) of the test item were added to 1 litre of dilution water and stirred for 24 h on a magnetic stirrer.
The pH was measured to be 8.0.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 25 mL and 0.111 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton stoppers.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
- Name : Desmodesmus subspicatus (formerly Scene-desmus subspicatus) Strain No. 86.81 SAG
- Source : Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Maintenance and Acclimatisation : Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21-24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- Preparation of pre cultures : Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
- Test cultures : The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium (annex 1) to make up to a final cell density of about 5000 cells per millilitre in the test medium.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
pH:
Control: 7.8 at 0 h and 7.9 at 72 h
Test items: 7.8 - 8.0 at 0 h and 7.8 - 7.9 at 72 h
Nominal and measured concentrations:
8 nominal concentrations (0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L active ingredient) plus control
The results are expressed in terms of geometric mean measured concentrations. Effective concentrations ranged from 98.1 % to 117.0 % of nominal values at 0 hours, and from 64.5 % to 90.3 % of nominal values at 72 hours.
Details on test conditions:
- Test vessels : 300 mL Erlenmeyer flasks with cotton stoppers, test volume: 25 mL
- Culturing apparatus : Climate chamber in which a temperature in the range of 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range of 400 to 700 nm. Temperature was measured and recorded daily in a water filled flask which was incubated under the same conditions as the test flasks.
- Light intensity : A light intensity ranging from 80 to 110 μE x m-2 x s-1, or an equivalent range of 5300 to 7300 lux, was measured. The light intensity was checked before the start of the test.
- Cell density measurements : Cell densities were measured in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced.
- Experimental design : 8 test concentrations plus 1 control, 3 replicates per concentration, 3 replicates per control, Initial cell density in the test cultures approximately 5000 cells per millilitre.
- Test item concentration/s : 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L active ingredient
- Method of administration : stock solution
- Duration of exposure: 72 hours
- Criteria of effects : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 32 - 61
Duration:
72 h
Dose descriptor:
other: ErC10
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 1.1 - 4.0
Duration:
72 h
Dose descriptor:
other: NOEC [r]*
Effect conc.:
>= 2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
* In this case NOEC/LOEC (72 h) determinations by statistical methods for growth rate does not appear to be meaningful. For growth rate (72 h) the statistical NOEC and LOEC values are 0.0285 mg/L and 0.0869 mg/L, respectively. In this case an effect of 6.0 % inhibition was determined as LOEC value, whereas from the biological point of view the result of only 6.0 % inhibition shows no biological effect. This is corroborated by various algal test guidelines (e.g. DIN 38 412 part 33) which consider only effects > 10 % as biologically relevant. In line with many regulatory-type decisions within the Existing Chemicals Programme of the EU, the EC10 values are generally regarded as surrogates for the NOEC. In the present case for growth rate the EC10 value of 2.4 mg/L should be used as a NOEC.
Validity criteria fulfilled:
yes
Remarks:
(-The factor of biomass parameter 131.0 >16; - The mean of the replicate coefficients of variation in the section-by-section growth 32.1 % <35 %; - The coefficient of variation of the mean specific growth rate replicates in the control 1.7 % <7%.)
Conclusions:
The toxicity to algae (Desmodesmus subspicatus) of the active ingredient of the substance was determined on the growth rate and showed an ErC50 of 43 mg/L and an ErC10 of 2.4 mg/L after 72 hours. A NOEC of >= 2.4 mg/L was calculated.
Executive summary:

In order to test acute toxicity to algae (Desmodesmus subspicatus) of the active ingredient of the substance was exposed to the test solution of eight nominal concentrations of the test item ( 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L) and blank control solution for a period of 72 hours under static conditions. The cell densities were measured at 24 hours intervals. Inhibition of the algal population was measured as reduction in growth rate (indexr), relative to control cultures grown under identical conditions. The measured concentartions confirmed that deviation from the nominal concentrations was less than 30 % ( Effective concentrations ranged from 98.1 % to 117.0 % of nominal values at 0 hours, and from 64.5 % to 90.3 % of nominal values at 72 hours). The growth rate was determined at an ErC50 of 43 mg/L and an ErC10 of 2.4 mg/L. A NOEC of >= 2.4 mg/L was calculated.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the acute algae study.

Description of key information

The toxicity to algae (Desmodesmus subspicatus) of the active ingredient of the substance was determined on the growth rate and showed an ErC50 of 43 mg/L and an ErC10 of 2.4 mg/L after 72 hours. A NOEC of 2.4 mg/L was calculated.

Key value for chemical safety assessment

EC50 for freshwater algae:
43 mg/L
EC10 or NOEC for freshwater algae:
2.4 mg/L

Additional information

In accordance with REACH Regulation, Art. 3, and ECHA "Guidance for identification and naming of substances under REACH and CLP" v1.2, March 2012, substances have to be separated from solvents if this is not affecting the stability of the substance or changing its composition.

In this case, separation of water would only be possible by thermical damaging of the substance. Furthermore, it was not possible to spray dry the pigment solution as it stuck to and partly melted on the conus dryer.