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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:
2012-02-28 till 2012-04-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling schedule: Concentrations of 6.3, 25 and 100 mg/L were measured at 0 and 72 hours; and 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:
To produce the different test item concentrations 6.1, 12.7, 24.7, 50.4 and 100.5 mg of the test item were weighed out and were added each to 1 litre of dilution water. All solutions were treated with an ultra turrax for 60 seconds at 8000 rpm and afterwards stirred for 24 h on magnetic stirrer. Undissolved particles of the test item were removed by filtration using folded filters with a pore size of 7-12 μm. The pH was measured and ranged between 7.6 and 7.8.

To produce the different test item concentrations 100 mL of each prepared solution was taken per replicate and 0.562 mL of the algal inoculum was added 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 / cellulose 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
pH:
Control: 7.9 at 0 h and 8.8 at 72 h
Test items:
6.3 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L and 100 mg/L : 7.7 - 7.8 at 0 h and 8.1 - 9.2 at 72 h
Nominal and measured concentrations:
6.3, 12.5, 25, 50 and 100 mg/L (nominal) plus control
Effective concentrations ranged from 95.6 % to 100.6 % of nominal values at 0 hours, and from 95.5 % to 101.4 % of nominal values at 72 hours.
Details on test conditions:
- Test vessels : 300 mL Erlenmeyer flasks with cotton / cellulose stoppers, test volume: 100 mL
- Culturing apparatus : Shaking incubator 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.
- 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 : 5 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. Additionally highest test concentration without algae.
- Test item concentrations : 6.3, 12.5, 25, 50 and 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:
other: ErC50
Effect conc.:
157.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence limits 132.4 - 312.1 mg/L
Duration:
72 h
Dose descriptor:
other: ErC10
Effect conc.:
87.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence limits 72.2 - 92.5 mg/L
Duration:
72 h
Dose descriptor:
other: NOEC [r]
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: LOEC[r]
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confindence limits 89.1 - 101.8 mg/L
Duration:
72 h
Dose descriptor:
other: EyC10
Effect conc.:
62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence limits 38.3 - 72-9 mg/L
Duration:
72 h
Dose descriptor:
other: NOEC[y]
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: LOEC[y]
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Remarks:
(- The factor of biomass parameter 137.0 >16 ;- The mean coefficient of variation for section-by-section specific growth 15.1 % <35 %; - The coefficient of variation of the mean specific grotwh rate replicates in the control 0.6 % <7 %)
Conclusions:
A growth rate of algae was measured at an ErC50 of 157.4 mg/L and a NOEC[r] at 100 mg/L was calculated (Neuhahn, 2012).
Executive summary:

In order to test acute toxicity of algae, Desmodesmus subspicatus, of the test substance were exposed to the test solution of five nominal concentrations of the test substance ( 6.3, 12.5, 25, 50 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. The measured concentrations confirmed that deviation from the nominal concentrations was less than 30 % (measured concentrations were in the range of 95.5 - 101.4 % of nominal concentrations). An ErC50 of 157.4 mg/L was determined and a NOEC[r] of 100 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

A growth rate of algae was measured at an ErC50 of 157.4 mg/L and a NOEC[r] at 100 mg/L was calculated (Neuhahn, 2012).

Key value for chemical safety assessment

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

Additional information

The test item consists of two components DEG-monophthalate and DEG-bisphthalate. The results for both individual components as well as its sum are reported.

In order to test acute toxicity of algae, Desmodesmus subspicatus, of the test substance were exposed to the test solution of five nominal concentrations of the test substance ( 6.3, 12.5, 25, 50 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. The measured concentrations confirmed that deviation from the nominal concentrations was less than 30 % (measured concentrations were in the range of 95.5 - 101.4 % of nominal concentrations). An ErC50 of 157.4 mg/L was determined and a NOEC[r] of 100 mg/L was calculated.