Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 May 2010 to 21 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with internationally recognised guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TFO
- Substance type:organic
- Physical state: liquid
- Lot/batch No.: TFOS285001
- Expiration date of the lot/batch: 30 June 2011
- Storage condition of test material: Room temperature

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.0954, 0.305, 0.977, 3.13, 10 mg/L

Samples at 0.0954 and 10 mg/L were analysed in the presence and in the absence of algal cells.


- Sampling method: complete contents of the sample bottle were analysed.

- Sample storage conditions before analysis: samples analysed immediately after being taken therefore no storage required. Duplicate samples were stored in a refrigerator in case further analysis was required.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: an accurate volume of the test substance was dispersed in sterile culture medium to prepare the highest test concentration of 100 mg/L. The remaining test solutions were prepared by further dilution of aliquots of the 100 mg/L solution.

- Controls: test medium

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata, Strain No. CCAP 278/4.
- Source: marine laboratory
- Method of cultivation:

Primary culture - Sterile algal nutrient medium (Appendix 2) was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C.

Secondary culture - appropriate volumes of the primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 7.82 x 105 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None

Test conditions

Test temperature:
21.6 to 22.2°C
pH:
7.4-9.90
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Control nd
3.13 mg/L 1.67 (0hrs) 1.53 (72 hrs) overall mean 1.60
10 mg/L 6.80 (0hrs) 6.38 (72 hrs) overall mean 6.59


The analaytical method was not sensitive enough to measure lower test concentrations therefore:

0.0954 to 0.977 mg/L, concentrations were calculated assuming 60.5% of their nominal value was present at 0 hours, and 56.5% of their nominal value at 72 hours; these values were based on the mean of the concentrations measured at the two highest levels at 0 and 72 hours.
Details on test conditions:
TEST SYSTEM
- Test vessel: Wheaton vials of approximately 65 mL capacity
- Type: closed
- Material, size, headspace, fill volume: glass, completely filled
- Aeration: gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at a nominal 130 cycles per minute.
- Initial cells density: 1 x 10E4 cells/mL.
- Control end cells density: 651700 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm).

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 4440 to 8880 lux (6 x 30 W “cool white” 1 metre fluorescent tubes)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Z Series Particle Count and Size Analyser.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3


- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study:
After 72 hours, algal growth was inhibited by ca 100% at 10 and 100 mg/L, by 12% at 1 mg/L and no significant inhibition occurred at 0.1 mg/L. The definitive test concentrations, selected based on the results of the range finding test, were 0.0954, 0.305, 0.977, 3.13 and 10 mg/L.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.966 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits, 0.736 & 1.21 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.37 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits, 2.09 & 2.72 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.179 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.572 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.22 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits, 1.85 & 2.70 mg/L
Duration:
72
Dose descriptor:
EC50
Effect conc.:
6.48 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits, 6.29 & 6.65 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.179 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.07 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: yield (biomass end-biomass start)
Remarks on result:
other: 95% confidence limits, 0.56 & 1.76 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.82 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: yield (biomass end-biomass start)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.179 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: yield (biomass end-biomass start)
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.572 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
other: yield (biomas start-biomass end)
Details on results:
- Exponential growth in the control (for algal test): yes/no

- Observation of abnormalities (for algal test): No microscopic abnormalities of the cells were detected.

- Any stimulation of growth found in any treatment: the levels of inhibition observed in average specific growth rate between 48 and 72 hours indicate some potential for recovery of the algal cells, as the higher test concentrations show a stimulatory effect.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium:
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Although a statistical NOEC could not be derived from the yield data, the inhibition observed at the two lowest concentrations (<15%) was not considered to be of biological significance. The EyC10 value, calculated as 1.07 mg/L, is considered to indicate the concentration at which algal growth was affected. Therefore, the NOEC and LOEC for yield were comparable with those calculated for the area under the growth curve and average specific growth rate; at
0.179 and 0.572 mg/L respectively.

The mean coefficient of variation (CoV) for daily growth rates in control cultures ranged between 2.55 and 10.2% during the definitive test and the CoV for the average specific growth rates of control cultures was 1.79% during the 72 hour exposure period.

Any other information on results incl. tables

It was not possible to develop a method of analysis with sufficient sensitivity to verify all of the exposure concentrations employed during the study (limit of detection of the analytical method defined as 0.5 mg/L). Therefore, to enable the test results to be expressed in terms of measured concentrations, the concentrations at 0.0954, 0.305 and 0.977 mg/L were calculated based on the measured levels of TFO at 3.13 and 10 mg/L.

At the start of the test, the measured levels in samples of the test cultures at 3.13 and 10 mg/L were 53% and 68% of their nominal values and after 72 hours, the measured levels were 49 and 64% of their nominal values, respectively. These results give overall mean concentrations of 0.0558, 0.179, 0.572 (calculated), 1.60 and 6.59 mg/L (measured). After 72 hours, it was not possible to detect any TFO in a sample of medium containing TFO at 0.0954 mg/L, which had been incubated without algal cells; however, in a sample at 10 mg/L, the measured level increased when compared to the initial concentration of TFO detected (83% of nominal compared to 68% of nominal). These results indicate that the concentrations of the test substance may have been influenced by the presence of algal cells.

The pH of the control cultures increased by more than 1.5 pH units during the 72-hour exposure period; the increase in pH observed during the definitive test is not thought to have affected either the validity or integrity of the study because the validity criteria for this type of test were met by the control cultures.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to TFO, the EbC50, ErC50 and EyC50 respectively were 0.572, 6.48 and 2.82 mg/L.

The “no observed effect concentration” (NOEC) for area under the growth curve, growth rate and yield was 0.179 mg/L.