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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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Intentional deviation in that the composition of the OECD medium and its pH were altered to suit the exposure regime employed. Reference the attached study report.
Principles of method if other than guideline:
Procedure 201 of the "Guidelines for Testing of Chemicals" of the Organisation for Economic Co-operation and
Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test, adopted 23 March 2006, Part C3 (Algal Inhibition Test) of the EC Methods for the Determination of Ecotoxicity: Annex to Directive 92/69/EEC O.J. L383A (29.12.92).
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Testing conducted prior to the definitive test indicated that losses due to volatility were only likely to occur whilst the test cultures were being shaken in the incubator. As a result, the test media were formulated without employing conditions for volatile chemicals. A concentrated solvent stock solution was made by dissolving the test substance (500 mg) in di-methylformamide (DMF, 10 ml) in a volumetric flask. This stock was serially diluted with
DMF to provide further stocks at nominal concentrations of 2.13, 4.67, 10.3 and 22.7 mg/ml. Aliquots (0.1 ml) of each solvent stock were added to algal culture medium (approximately 700 ml) in volumetric flasks. The contents of each flask were shaken vigorously then treated by ultrasound (30 minutes) before being made up to volume (1000 ml) with sterile culture medium. The contents of each vessel were stirred overnight and left to stand for 10 minutes before being used in the test. An aliquot (5.30 ml) of the secondary algal inoculum was added to a portion (800 ml) of the test medium at each concentration, to give an initial cell density of 1 x 104 cells/ml. Aliquots (65 ml) of the appropriate inoculated test medium were added to each of the test vessels, which were completely filled with no headspace and sealed.

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
yes
Details on test solutions:
Dimethylformamide (DMF)

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokircheriella subcapitata, Strain no. CCAP 278/4

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
After 72 hours of exposure to Trimofix O, the EbC50 was estimated to be > 2.93 mg/l (36% inhibition)
Post exposure observation period:
After 72 hours of exposure to Trimofix O, the EbC50 was estimated to be > 2.93 mg/l (36% inhibition). An EbC50 value of 3.09 mg/l (95% confidence limits, 3.01 and 4.12 mg/l) was calculated by extrapolation of the logistic curve.

Test conditions

Hardness:
Hardness was controlled by the addition of macro-nutrients noted in Appedix 2 of the Attached report.
Test temperature:
Temperatures of the controls & test solutions at 0h and 72 h are outlined in Table 5 (a) of the Attached report.
pH:
pH's of the controls & test solutions at 0h and 72 h are outlined in Table 5 (a) of the Attached report.
Dissolved oxygen:
Not measured.
Salinity:
Not Applicable
Nominal and measured concentrations:
Reference Table 1 of the Attached report.
Details on test conditions:
Test vessels each containing control or test culture (65 ml) were placed in an illuminated orbital incubator according to a random number sequence; their position in the incubator was re-randomised after 24 and 48 hours. The cultures were incubated, without renewal of medium for 72 hours under continuous illumination provided by 30 W “cool white” 1 metre fluorescent tubes. The light intensity measured from the four extremities and one central position in the incubator on each occasion (0, 24, 48 and 72 hours) ranged from 5750 to 7820 lux, with a mean value of 6872 lux. The minimum and maximum light intensity on each occasion varied from the mean by a maximum of –16 and +12% over the test period (see protocol deviations). The temperature was maintained at 22.0 to 23.9°C. Temperature and pH of control and test flasks at the start and end of the test were recorded.
Although the pH of the control medium increased by more than 1.5 units; this does not invalidate the test as similar increases in pH were observed across all test groups and the objectives of the study were met. Suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at a nominal 150 cycles per minute.
Reference substance (positive control):
no
Remarks:
Control & Solvent Control Included

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 3.38 & 7.40 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.09 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL 3.01 & 4.12 mg/l
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.841 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.841 mg/L
Basis for effect:
biomass
Details on results:
The results of chemical analysis are given in Table 1 and example chromatograms are illustrated in Appendix 3. At the start of the test, the measured levels of Trimofix O in samples of the test cultures ranged between 68 and 78% of their nominal values except at 2.27 mg/l where the measured
level was 44% of its nominal value. Failure to achieve the nominal concentrations was attributed to the low aqueous solubility of the test substance (5 mg/l). After 72 hours, the measured levels had decreased, ranging from 66 to 74% of their initial values giving overall measured levels of 0.132, 0.282, 0.650, 0.841 and 2.93 mg/l, based on a geometric mean. After 72 hours, analysis of samples of medium containing Trimofix O at 0.213 mg/l and
5 mg/l, which had been incubated without algal cells, indicated that the test concentrations were maintained at 84 to 92% of their initial starting values respectively. These results suggest that the loss of the test substance observed during the study was due to the presence of algal cells.
Results with reference substance (positive control):
Not applicable

Applicant's summary and conclusion

Conclusions:
After 72 hours of exposure to Trimofix O, the EbC50 was estimated to be >2.93 mg/l (36% inhibition). An EbC50 value of 3.09 mg/l (95% confidence limits, 3.01 and 4.12 mg/l) was calculated by extrapolation of the logistic curve. The ErC50, was estimated to be >2.93 mg/l (7% inhibition). An ErC50 value of 3.60 mg/l (95% confidence limits, 3.38 and 7.40 mg/l) was calculated by extrapolation of the logistic curve. The “no observed effect concentration” (NOEC) for area under the growth curve and growth rate was 0.841 mg/l.
Executive summary:

After 72 hours of exposure to Trimofix O, the EbC50 was estimated to be >2.93 mg/l (36% inhibition). An EbC50 value of 3.09 mg/l (95% confidence limits, 3.01 and 4.12 mg/l) was calculated by extrapolation of the logistic curve. The ErC50, was estimated to be >2.93 mg/l (7% inhibition). An ErC50 value of 3.60 mg/l (95% confidence limits, 3.38 and 7.40 mg/l) was calculated by extrapolation of the logistic curve. The “no observed effect concentration” (NOEC) for area under the growth curve and growth rate was 0.841 mg/l.