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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to protocol.
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
no chemical analyses have been performed; WAF method was used; The amount of NaHCO3 was elevated to assist pH stability during the test; Biomass (dw/mL) was not determined at the end of the test in the control vessels.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on test solutions:
Preparation of solutions
A WAF was prepared for the test concentration by adding of an accurate amount of the test substance weighed on an analytical balance vastly in excess of the water solubility limit to the test media. This solution was allowed to equilibrate under slow-moderate agitation over 24 hours in sealed 1 L glass vessels and then stand for approximately an hour before use. Each WAF was then considered loaded with the test substance components and / or breakdown products at their corresponding water solubility limits and was transferred without solid test material or film to the test vessels. The test vessels were filled with 40 mL of the corresponding WAF test solution or control. The test concentration was tested in triplicate prepared from the same WAF. The inoculum was then added to the vessel from an exponentially growing culture and the test vessel was sealed with a cotton wool stopper and incubated for the test duration. In addition, 6 control replicates were tested containing test media only. The extinction of the contents of each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours.
A 24 hour stirring period was used based on a series of non GLP preliminary tests investigating extended stirring periods.

Test concentrations
One WAF loaded with a 100 mg of test substance and one control containing only test medium were made as previously described and used directly for testing.
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, Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK. After purchasing, this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase.
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Test temperature:
23.05 - 23.8 °C
8.0 - 10.3
Details on test conditions:
- Test vessel: 100 mL Erlenmeyer flasks
- Type (delete if not applicable): open, closed with a cotton wool stopper
- Aeration: no
- Initial cells density: 1x10^4 cells/mL
- Control mean specific growth rate: 0.077
- 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: contiuous light
- Light intensity and quality: 104.7 and 104.2 μmol/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

- Range finding study
A series of preliminary tests were conducted using identical methods and materials as detailed for the definitive test but with varying amounts of time for stirring of the WAF preparations. WAF preparations were made at 100 mg/L loading and stirred for 24, 48, 72 and 2 weeks. No toxic effects on the test organisms were observed. A 24 hour stirring period with 100 mg/L loading was therefore used in the definitive test.
Reference substance (positive control):
potassium dichromate
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: loading rate
72 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: loading rate
Details on results:
Statistical comparison of the test replicates with the control showed no statistically significant differences.

The pH measurements show a maximum increase of 2.1 pH units in the control replicates which exceeds the guideline recommendation of 1.5. This is however easily explained by the control growth that was slightly higher than usually achieved in 72 hours.

Three control cultures and two test replicates were inspected microscopically at the end of the test and no significant bacterial contamination was observed.
Results with reference substance (positive control):
For the evaluation of the quality of the algae and the experimental conditions, the reference substance potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions and algae sensitivity.
Reported statistics and error estimates:
Was conducted using the Toxcalc 5.0 software package and Wilcoxons two sample test to check for significant difference in comparison to the control.
Validity criteria fulfilled:
Not possible to determine test chemical stability. A large excess of the test chemical was loaded to the test media in the WAF to ensure stabilization at the water solubility limit even if degradation of the test chemical was to occur.
This study is reliable without restrictions. It is performed according to OECD guideline 201 under GLP and all critical validity criteria were fulfilled.
The test substance concentration was not monitored during the test as chemical analysis was technically not feasible. The calculated endpoints are adequate for C&L and risk assessment purposes.

Considering the ready biodegradability and lack of detectable toxicity the test substance is not expected to present a hazard to algae in the aquatic environment.
Executive summary:

In order to predict the effects of Dihexadecyl peroxodicarbonate in an aquatic environment, an Algal Growth Inhibition test was conducted in accordance with OECD test guidelines and with the OECD Principles of Good Laboratory Practice. Some minor modifications to the guideline were applied due to inherent properties of the test chemical. These included preparing the test chemical as a Water Accommodated Fraction (WAF), minor changes in media components to help aid pH stability.

The toxicity of the test chemical to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. A nominal loading concentration of 100 mg/L as a limit test was conducted.

No significant statistical difference in growth rate or biomass was detected in comparison to the control. The test substance was concluded as not toxic to algae at its solubility limit in the test medium.

The following quality criteria have been met in the present study:

- The cell density of the controls increased at least a factor 16 within 72 hours.

- The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7%.

- The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L.

- The mean coefficient of variation for section-by-section specific growth rates in the control did not exceed 35 %.

The following criteria could not be determined:

- The stability of the test chemical during the test period. Chemical analysis was concluded as technically not feasible after a series of preliminary tests and was therefore not conducted during the study. The WAF solution was loaded with a significant excess of test chemical and preparation times were investigated prior to the study to ensure a worst case scenario was tested in the absence of supporting analytics.

Considering the ready biodegradability and lack of detectable toxicity the test substance is not expected to present a hazard to algae in the aquatic environment.

Description of key information

Considering the ready biodegradability and lack of detectable toxicity the test substance is not expected to present a hazard to algae in the aquatic environment.

Key value for chemical safety assessment

Additional information