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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
GLP study according to protocol. The CoA included in the report did not contain enough detail on composition; a statement of the composition is attached to the robust summary. No detailed description of the results of the chemical analyses and system stability.
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
The NaHCO3 concentration in the test medium was 150 mg/L.
GLP compliance:
Analytical monitoring:
Details on sampling:
At the beginning of the test, after 24 hours and at the end of the test, samples were taken from the control, lowest, middle and highest test concentration for chemical analyses. At each sampling time and for each concentration, 1.6 ml of sample was taken from each of the three flasks with same concentration and were pooled. After filtration to remove the algae using a Millex GP 0.22 µm filter the pooled sample was immediately subjected to the chemical analyses.
Details on test solutions:
The test substance is soluble in water. A stock solution of approximately 1 g/L of test substance was prepared as follows: an accurately measured amount of 0.5064 g. of test substance was weighed into an 500 ml volumetric flask and 400 ml of deionised water was added and concentrated OECD medium stock solutions enough for 500 ml solution. After homogenization, the pH of the solution was checked and adjusted to 7.9 using a 1 M NaOH solution. Then the solution was filled up to the 500 ml line on the flask with deionised water.

The test solutions were prepared by addition of adequate amounts of stock solution to the test vessels. The following nominal test concentrations were prepared: 4.30, 9.37, 20.76,45.58 and 100.01 mg/L in test medium. Controls containing only test medium were included in the test
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out with the freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, The Windermere Laboratory, Cumbria, Ambleside, United Kingdom. After purchasing this strain was cultured and
maintained according to Standard Operation Procedures. Cultures on sloped agar tubes were stored at 4°C until required.

The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 ml) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing.
The extinction of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1E04 cells/ml in the test medium.
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Test temperature:
22.6 to 23.6°C
Nominal and measured concentrations:
Nominal test material: 4.30, 9.37, 20.76, 45.58 and 100.01 mg/L
Time weighted average test material: 3.6, -, 15.0, -, 62.2 mg/L
Time weighted average active ingredient: 1.1, -, 4.5, -,18.6 mg/L
Details on test conditions:
- Test vessel: 100 mL Erlenmeyer flasks, with 40 mL of test medium
- Type (delete if not applicable): open, stoppered with a cotton wool stopper
- Aeration: no
- Initial cells density: 1*10^4 cells/mL
- Control end cells density: 1.34*10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Standard medium used: yes, OECD medium

- Source/preparation of dilution water: OECD medium
- Culture medium different from test medium: no

- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 96 and 98 µmol/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer
Reference substance (positive control):
Potassium dichromate
72 h
Dose descriptor:
Effect conc.:
3.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Recalculated by the reviewer. EC10 not given in the report
72 h
Dose descriptor:
Effect conc.:
5.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Recalculated by the reviewer. EC50 reported as measured initial test material concentration.
Details on results:
Remark from the reviewer:
No EC10 was mentioned in the report, this endpoint has been calculated in TOXCALC v5.023 with the raw data given in the report and added to this robust summary by the reviewer.
The results given in the report were recalculated to time weighted average concentration of the active ingredient instead of measured initial test material concentrations as the test material was not stable during the test. The results mentioned in the section on Effect concentrations are the values converted to time weighted average of the active ingredient.

Analytical results:
the initial measured concentrations are between 11 to 18% lower than the nominal calculated concentration. Furthermore, it is clear that the test substance is not stable under test conditions. The initial measured concentration of the parent compound decreases up to almost 50% during the test at the highest test concentration.
Reported statistics and error estimates:
The mean values of the extinction's for each test substance concentration were used to calculate the growth and specific growth rate.
Where possible, the EC20,50,80 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance. The EC50 value calculated for the area under the growth curve is termed EbC50 (0-72 h), whereas the EC50 value calculated for the specific growth rate is termed ErC50.
The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the growth at each concentration and the control using threshold values from the William's test. The No Observed Effect Concentration (NOEC) was derived from the results as the first concentration below the LOEC value, where growth shows no significant inhibition relative to the control values. Confidence limits were computed on the basis of Fieller's theorem. All computations were performed using the TOXCALCTM version 5.0 program.

The revised OECD guideline of 2006 contains some additional validity criteria. These criteria have been verified for this study and are found to be within the limits given in the guideline. See for details the attached tables.

Validity criteria fulfilled:
This study is reliable with the above mentioned restrictions. It is performed according to OECD guideline 201 under GLP and all validity criteria were fulfilled.
The test substance was not stable during the test and therefore measured TWA active ingredient concentrations were used. The calculated endpoints are adequate for C&L and risk assessment purposes.
Executive summary:

In order to predict effects of chemicals in an aquatic environment, the toxicity to freshwater algae was assessed. The algal toxicity was determined in the Algal Growth Inhibition test in accordance with OECD, EEC and ISO test guidelines and with the OECD Principles of Good Laboratory Practice. The guidelines were slightly modified to ensure good growth and pH control of the cultures.

The green algae Pseudokirchneriella subcapitata (formerly known as Selenastrum capricomutum) was exposed to the following concentrations: 4.30, 9.37, 20.76, 45.58 and 100.01 mg/L of acetyl acetone peroxide in test medium.

The chemical analyses performed on the samples taken during the test demonstrated that the test substance is not stable during the test. At the end of the test only 52% of the initial concentration was found at the highest test concentration. Despite the instability of the parent compound, the effects are all calculated based on the concentration of parent compound as measured at the beginning of the test.

The toxicity of acetyl acetone peroxide to exponentially growing culture of Pseudokirchneriella subcapitata was determined over an exposure period of 72 hours. The EbC50 and ErC50 (0-72 h) values of the test substance as calculated from the dosis effect curve for P. subcapitata are 18.71 mg/l and 27.91 mg/L (22.10 - 36.78 mg/l, 95% confidence limits), respectively. The NOEC determined from the results is 9.37 mg/L, the LOEC is 20.76 mg/L. The test was conducted in a mineral salts medium in a climatized illuminated orbital incubator. The maximum variation in pH in the test media was 0.8 pH unit.

The definitive test is valid as shown by the increase of the extinction of the control over 72 h by a factor of 134.

Remark from the reviewer: The results given in this summary are as given in the report, so have not been converted to TWA active ingredient concentrations.

Description of key information

The 72h-ErC10 for freshwater algae is 3.5 mg a.i./L (measured. TWA) and the 72h-ErC50 is 5.4 mg a.i./L (measured TWA)

Key value for chemical safety assessment

EC50 for freshwater algae:
5.4 mg/L
EC10 or NOEC for freshwater algae:
3.5 mg/L

Additional information