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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)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed ptotocols, with no deviations from standard test guidelines and no methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Lot number : 1-022
Appearance : colorless liquid
Assay= 98.84 %
Acidity (HBr) < 0.01 %
Container: glass flask
Storage conditions: at room temperature

Sampling and analysis

Details on sampling:
After 24, 48 and 72 h , a sample of 5 ml wa taken.

Test solutions

Details on test solutions:
The stock solutions, for the preliminary and definitive tests, were prepared by dispersing the test item directly in LC reconstitued water.
The test method was adapted to volatile substances since a significant loss of Bromure de n-amayle from the test solutions by volatilization was possible during preparation of these solutions and during the tests. Test vessels were sealed and 0.3 g/l of NaFCO3 and 6 mM of HEPES buffer were added to LC reconstitued water (to prevent limitation of growth coming from CO2depletion and important increasing pH).

Preliminary test
A loaing rate of 100 mg/l was used to prepare the stock solution. The stock solution was agitated for 23 hours in a closed flask (headspace kept at the minimum) and filtered through a filter of porosity 0.45 µm to remove the suspended fraction. The filtered solution, considered as the limit of solubility of the test item under our experimental conditions 5LS), was then used to prepare the test solutions.

-Limit test
Three replicate solutions of algae with an initial dilution of 10E4 cells/ml were exposed to the LS solution (the limit test solution) and a further group of six replicate algal solutions without test item was used as the control. The growth of the cell culture in each flask was monitored by counting the number of cells at 24, 48 and 72 hours.
- Range-finding test
Three groups of two replicate flask were xposed to dilutions of the limit test solution at LS/1000, LS/100 and LS/10 under the same conditions of light and temperature.

Definitive test
A loaing rate of 500 mg/l was used to prepare the stock solution. The stock solution was agitated for 23 hours in a closed flask (headspace kept at the minimum) and filtered through a filter of porosity 0.45 µm to remove the suspended fraction. The filtered solution, considered as the limit of solubility of the test item under our experimental conditions 5LS), was then used to prepare the test solutions.
Six concentrations of Bromure de n-amyle were used together with a control at: 0 mg/l, LS/32, LS/16, LS/8, LS/4, LS/2 and LS.
Three replicates were exposed to each concentration and six replicates to dilution water only (to act as the control) for 72 hours. Observations of cell growth were recorded at 24, 48 and 72 hours.


Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Strain CCAP 278/4
Obtained from Culture Center of Algae and Protozoa
The algae are cultured under sterile conditions and maintained at exponential growth rate.

Study design

Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Post exposure observation period:
Not done

Test conditions

Hardness:
34 ± 17 mg/l as CaCO3
Test temperature:
Between 22.4 to 25.4°C
pH:
Between 7.20 and 7.50 at the beginning and between 7.28 and 7.39 at the end of the study (72 hours)
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
At the beginning of the test, measured concentrations were 0.618, 1.76, 3.92, 12.0 and 41.1 mg/l at LS/16, LS/8, LS/4, LS/2 and LS respectively. The actual concentration was lower than the limit of quantification (0.5 mg/l) at LS/32.
Details on test conditions:
Test flasks were incubated at 23±2°C continuously agitated and constantly illuminated.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate, purity > 99.95 %

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
55.5 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 35.4 -128 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.85 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL = 7.15 - 11.2 mg/l
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.76 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: not determined
Details on results:
The ErC50 72-hour was estimated by extrapolation since the inhibition percentage of the growth rate at the highest concentration (41.1 mg/l) was slightly lower than 50% at 72 hours (45%).
Results with reference substance (positive control):
The validity criteria of the study were respected: the increase in cell density (R), measured in the control solution between the end and the beginning of the test, was greater than a factor of 16 (R = 105) ; the final concentrations of ethyl bromide were maintained within the designated limit of 80% of the initial concentrations in non-inoculated flasks.
Reported statistics and error estimates:
After checking of the normality of the data Chi-square and Shapiro-Wilks test (normal at p = 0.01) as well as the variance homogeneity (Bartlett test), the NOEC is determined by ANOVA (the Bonferroni T-test) using the individual replicates of the area under the curve and the specific growth rate.

Any other information on results incl. tables

The test solution was a clear and colorless solution at all test item concentrations.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, toxicity to aquatic algae of the test material was determined to be 10 < ErC50 < 100 mg/l.
It has been to classify as Dangerous for environment Tox 2
Executive summary:

Under the experimental conditions, the 72-hour ErC50 and the 72-hour EbC50 of Bromure de n-amyle in a static test system were estimated as 55.5 and 8.85 mg/l (based on initial measured concentrations in test solutions), respectively, for Pseudokirchnerella subcapitata.

The NOEC at 72 hours was 1.76 mg/l based on the specific growth rate and biomass data.

It has been to be classify as Dangerous for environment Tox 3 according to Regulation 1272/2008.