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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:
2011-07-18 to 2011-08-12
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling Schedule: Concentration of 100 mg/L was measured at 0 and 72 h; and concentration of 0 mg/L at 72 h 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:
To produce the only test item concentration 100.1 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 a folded filter with a pore size of 7 - 12 μm.
The pH was measured to be 8.8 and was adjusted to pH 7.7.
100 mL of the solution were taken and 0.806 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 to make up to a final cell density of about 5000 cells per mL in the test medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21 - 24 °C
pH:
Control: 7.7 at 0 h and 9.1 at 72 h
100 mg/L: 7.7 at 0 h and 8.0 at 72 h
Nominal and measured concentrations:
The test concentrations were measured at 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominal) and control
The lowest test item concentration 0.1 mg/L slightly dropped below the 80 % recovery limit during the 72-hour exposure period. This is considered as not significant, because the higher test item concentrations remained stable over the exposure time and therefore all results are expressed in terms of nominal concentrations. Effective concentrations ranged from 94.0 % to 99.1 % of nominal values at 0 hours, and from 70.0 % to 99.6 % of nominal values at 72 hours.
Details on test 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 : 7 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 : 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
- 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
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
18.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 16.0 – 22.1
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
2.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 1.8 – 3.8
Duration:
72 h
Dose descriptor:
other: NOEC [y]
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Validity criteria fulfilled:
yes
Remarks:
-The factor of biomass parameter 110.0 >16; -The mean of the replicate coefficients of variation in the section-by-section growth rate was: 34.5 % <35 %; -The coefficient of variation of the mean specific growth rate replicates in the control 1.8 % <7 %.
Conclusions:
A 72-h growth inhibition test with the algae Desmodesmus subspicatus was performed with the test substance dibenzylamine according to OECD guideline 201 (GLP conditions). It can be concluded that the test item had no significant inhibitory effect on the growth of Desmodesmus subspicatus up to and including the test concentration of 0.1 mg/L, after the test period of 72 hours. The EC50 for growth rate inhibition (72-h ErC50) was 18.8 mg/L with a 95% confidence interval ranging from 16 to 22.1 mg/L. The results of the test can be considered reliable without restrictions.
Executive summary:

In order to test acute toxicity to algae of the substance, Desmodesmus subspicatus was exposed to 7 nominal concentrations of the test substance (0.1, 0.32, 1, 3.2, 10, 32 and 100 mg/L) and blank control solution for a period of 72 h under static conditions.

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. Growth rates were also used to calculate a "No Observed Effect Concentration" and a "Lowest Observed Effect Concentration" according to Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment. The following values were determined: ErC 50* (0-72 h): 18.8 mg/L, ErC 10* (0-72 h): 3.2 mg/L, NOEC [r] (tα 0.05): 0.1 mg/L and LOEC [r] (tα 0.05): 0.32 mg/L.

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

Description of key information

The study of Currenta (2011), investigating the acute toxicity of dibenzylamine to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. The 72-h NOEC and 72-h EC50 for growth rate were determined to be 0.1 mg/L and 18.8 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
18.8 mg/L
EC10 or NOEC for freshwater algae:
0.1 mg/L

Additional information

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. Growth rates were also used to calculate a "No Observed Effect Concentration" and a "Lowest Observed Effect Concentration" according to Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment.