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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Directive 92/69/EEC, C.3, 1992
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal 100 mg/L and control
- Sampling intervals: start (0 h), and at test end (72 h)
- Sampling method:
At test start: unfiltrated and filtrated (maximum pore size approx. 1.2 µm) samples from the freshly prepared undiluted supersaturated suspension.

At test end: unfiltrated and filtrated (maximum pore size approx. 1.2 µm) samples from undiluted supersaturated suspensions, incubated in parallel without algae under the same conditions as in the actual test were collected (collecting of representative suspension-samples from the actual test itself was not possible since the test item was not homogeneously distributed in the test medium).
Control samples were also incubated in parallel.
Samples were taken in duplicate.
- Sample storage conditions before analysis: The filtrated samples were deep-frozen (at about -20 °C) immediately after sampling, all the other samples were analyzed immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item the supersaturated suspension with a nominal concentration of 100 mg/I was prepared as follows: 300 mg of the test item were suspended in a small volume of test water by using a homogenisator. Then the mixture was transferred quantitatively into an adequate volume of test water to obtain a final concentration of nominal 100 mg/L. This suspension was treated ultrasonically for 15 minutes. Then, the supersaturated suspension was stirred by a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum concentration of the test item in the suspension. No auxiliary solvent or emulsifier was used. The stirring period of 96 hours was chosen according to the results of a pre-test, which showed that during this time no significant loss of the test item was to be expected. After stirring and after another 15 minutes ultrasonic treatment prior to the start of the test, a homogeneous suspension was obtained. The undiluted supersaturated suspension with the nominal concentration of 100 mg/L dissolved and suspended test item was used as test medium.
- Controls: Test water without addition of the test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
At the start of the test, the test item was very finely and homogeneously suspended in the test medium. After about one hour, test item particles were observed swimming at the water surface. After one day, a part of the test item was swimming at the water surface and was lying on the bottom of the test beaker. The test flasks, however, were continuously stirred.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: 86.81 SAG
- Source (laboratory, culture collection): SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).

- Age of inoculum (at test initiation): An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and kept in the exponential growth phase until inoculation of the test solutions.
- Method of cultivation: standardized conditions according to the test guidelines

ACCLIMATION
- Acclimation period: three days (pre-culturing)
- Culturing media and conditions (same as test or not): same
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.24 mmol/l as CaCO3
Test temperature:
23°C
pH:
7.8 - 8.9
Nominal and measured concentrations:
Nominal and measured concentrations
nominal loading rate: 100 mg/L
measured test concentrations
- unfiltered sample of the suspensions: 83% and 89% of the nominal value (total mean 86%)
- dissolved and very finely suspended test item (measured in the filtrate of the samples): below the limit of detection (LOD) of approx. 1.0 mg/L.

The biological results are based on the loading rate (nominal concentration) of the test item.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks containing 15 mL test medium, covered with a glass lid
- Aeration: continuous stirring by magnetic stirrers
- Initial cells density: 10'000 cells/mL
- Control end cells density: 117.0 x 10E4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per concentration without algae (replicates): 2 (particle controls; see below)
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (acc. to OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The light intensity was measured at the start of the test. The pH was measured and recorded in each treatment at the start and end of the test. The water temperature was measured at least daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test medium was recorded at least daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination by fluorescent tubes (Philips TLD 36W/840), installed above the water bath in a distance of about 35 cm from the test flasks
- Light intensity and quality: The light intensity at the level of the test solutions was about 7000 Lux (range: 7500 – 8000 Lux), measured at six places distributed over the experimental area at the surface of the test media). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : daily determination of the algal biomass (cell counts)
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
electronic particle counter (Coulter Counter®, Model ZM). Counted algal cell densities were corrected for the particle control (supersaturated suspension with a nominal concentration of 100 mg/L without algae). The measurements were performed at least in duplicate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)

- Range finding study
- Results used to determine the conditions for the definitive study: the test concentration was based on the results of a range-finding test and the results of pre-experiments to the solubility of the test material.
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: growth rate and biomass
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: growth rate and biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: no significant stimulation of growth (see attached document "Tables Cell densities and inhibition of growth.pdf")
- Effect concentrations exceeding solubility of substance in test medium: Yes; the solubility of the test item in test water was below the LOQ of the analytical method (< 1 mg/L).

For details see attached document "Tables Cell densities and inhibition of growth.pdf"
Results with reference substance (positive control):
not reported
Reported statistics and error estimates:
not applicable
Validity criteria fulfilled:
yes
Conclusions:
The results indicate that the test material is not toxic to algae up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.

Since no classification criteria according to GHS/CLP (Regulation (EC) No. 1272/2008) are fulfilled, the substance is not classified for environmental hazards.
Executive summary:

Executive summary


Due to the low water solubilty of the test item, an unfiltrated supersaturated suspension with a nominal concentration of 100 mg/l was used as the test medium, containing a max. concentration of the test substance.


During the test period the total mean measured concentration of dissolved and suspended test substance in the unfiltrated duplicate samples of the suspension was 86% of the nominal value. Under the test conditions, the concentration was sufficiently constant during the 72-hours test period. The concentration of dissolved and very finely suspended test substance was below the detection limit of approx. 1.0 mg/l.


As no effect on biomass and algal growth was observed, the 72-hour EL10 (NOELR) and thus a PNEC cannot be determined.

Description of key information

effect values for Desmodesmus subspicatus, based on loading rates (OECD 201, EU C.3):
0-72 h average specific growth rates:
EL50: >100 mg/L, EL10: >100 mg/L; NOELR: >100 mg/L (solubility in test water < 1 mg/L)

Key value for chemical safety assessment

Additional information

EL50 for freshwater algae: > 100 mg/L
EL10 or NOEL for freshwater algae: > 100 mg/L


The results indicate that the test material is not toxic to algae up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.

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