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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
Study period:
2011-03-17 till 2011-05-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Sampling schedule:
Control at 72 hours
Test concentration (100 mg/L) at 0 and 72 hours
Vehicle:
no
Details on test solutions:
To produce the only test item concentration 100.4 mg of the test item were added to 1 litre of dilution water and treated for one minute at 8000 rpm with an ultra turrax and afterwards stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using an aseptic filter, Sartobran 150, with a pore size of 0.45+0.2 μm. The pH was measured to be 7.5.

100 mL of the solution were taken and 0.555 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 6 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 Scenedesmus 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 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
Test temperature:
21 - 24 °C measured at each test vessel at the beginning and the end of the test.
pH:
7.6 - 9.7 measured at each test vessel at the beginning and the end of the test.
The pH value in the test item slightly increased by more than 1.5 pH units. This increase is not regarded to be relevant to the results as all validity criteria were met.
Nominal and measured concentrations:
100 mg/L (nominal) plus control
Details on test conditions:
Exposure conditions
- Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers; test volume: 100 mL
- Culturing apparatus: Light chamber in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 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 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light
intensity was checked before the start of the study.
- 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: 1 test concentration plus 1 control; 6 replicates per concentration/control; initial cell density in the test cultures approximately 5000 cells per millilitre; additionally highest test concentration without algae.
- Test item concentration: 100 mg/L
- Method of administration: direct weighing
- 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:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: LOEL
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The substance is insoluble or poorly soluble in water. Therefore a suitable selective and sensitive chromatographic method for the determination of the test item in aqueous solutions could not be established.
The results are expressed in terms of Effective Loadings (EL). As the test item is a multi constituent and no information about the correlation between molecular weight and the structural formula of the test item are available, a Water Accommodated Fraction (WAF) was used to test effects at a limit concentration of 100 mg/L, and no specific analysis was performed. With the sponsor’s agreement, the content of the test item during the exposure period was verified by DOC determination.

An analysis of the yield and growth rate of the algal population within the 72 h exposure period gave the following results:

Results [mg/L]:

ErL 50 (0-72 h): > 100

ErL 10 (0-72 h): > 100

NOEL [r] (tα 0.05): ≥ 100

LOEL [r] (tα 0.05): > 100

EyL 50 (0-72 h): > 100

EyL 10 (0-72 h): > 100

NOEL [y] (tα 0.05): ≥ 100

LOEL [y] (tα 0.05): > 100

No toxic effects against algae were observed at a limit test concentration of 100 mg/L.

Remark:

Reduction of growth rate (ErLx, NOEL [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyLx, NOEL [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU) and are given in the results section of this report.

Validity criteria fulfilled:
yes
Remarks:
factor of biomass parameter 91.7 > 16 within 72 h; mean of the replicate coefficients of variation in the section-by-section growth rate was 11.3 % < 35 %; coefficient of variation of the mean specific growth rate replicates in the control was 1.4 % < 7 %
Conclusions:
No toxic effects against algae were observed at a limit test concentration of 100 mg/L of Castor Oil, reaction product with Soybean Oil.
Executive summary:

A study was performed to assess the adverse effects of the substance on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 "Freshwater Alga and Cyanobacteria, Growth inhibition test" (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006). Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of the substance dissolved in dilution water. Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer and an aseptic filter. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. After 72 hours ErL50 of > 100 mg/L, ErL10 of >100 mg/L, NOEL [r] of ≥100 mg/L and a LOEL [r] of >100 mg/L were determined. The substance is insoluble or poorly soluble in water. Therefore a suitable selective and sensitive chromatographic method for the determination of the test item in aqueous solutions could not be established. The results are expressed in terms of Effective Loadings (EL). As the test item is a multi constituent and no information about the correlation between molecular weight and the structural formula of the test item are available, a Water Accommodated Fraction (WAF) was used to test effects at a limit concentration of 100 mg/L, and no specific analysis was performed. With the sponsor’s agreement, the content of the test item during the exposure period was verified by DOC determination.

Description of key information

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 "Freshwater Alga and Cyanobacteria, Growth inhibition test" (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006) to assess the adverse effects of the substance, on the growth rate of algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus). Algal cells were exposed for 72 hours to a limit test concentration of nominally 100 mg/L of the substance dissolved in dilution water. After 72 hours ErL50 of >100 mg/L, ErL10 of >100 mg/L, NOEL [r] of ≥100 mg/L and a LOEL [r] of >100 mg/L were calculated. The results are expressed in terms of Effective Loadings (EL).

There was no effect at an exposure concentration of 100 mg/L in this limit test.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

The key value should read ErC50 > 100 mg/L and ErC10 > 100 mg/L as there was no effect in this limit test.