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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
Study period:
26 March 2007 to 29 March 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study undertaken by Japanese accredited laboratory to internationally recognised guidelines.
Qualifier:
according to
Guideline:
other: Heisei 15/11/21-No.1·121002 of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare
Deviations:
no
Qualifier:
according to
Guideline:
other: Heisei 15/11/13-No.2 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry
Deviations:
no
Qualifier:
according to
Guideline:
other: No.031121002 of the Environmental Policy Bureau, Ministry of the Environment, Japan
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test substance was added to the growth media. Through 48 hours mixing by magnetic stirrer, in the dark place, at 23 °C ± 2 °C, original solution of the test substance (100 mg/L) was prepared. However, there were small amount of undissolved test substance in the original solution of the test substance. These were removed by filtration with glass fiber filter (GF/F [Whatman International Ltd.]). The test solutions for each concentration groups were prepared by adding the original solution of the test substance by aseptic manipulation to the growth media for the test or direct usage. Note that the growth media for the test at the time of preparation of the test solutions were used at 23 °C ± 2 °C by adjusting beforehand.
Note that, since the purity (95.9 %) was not considered for amount of the test substance, the nominal concentrations for each group were indicated in the concentration of the supplied substance.


Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC22662
- Source: American Type Culture Collection
- Method of cultivation: cultured under aseptic conditions

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23.3 - 24.0°C
pH:
7.7 – 8.0 (pH control of solution is not applied)
Nominal and measured concentrations:
Nominal: 4.6, 10, 22, 46 and 100 mg/L
Measured: 3.4, 8.5, 19.6, 44.7and 88.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL volume conical glass flasks (with air-permeable silicon stopper)
- Type: closed
- Fill volume: 100 mL
- Aeration: none
- Type of flow-through: static
- Initial cells density: 1 x E+04 cells / mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: 80 - 82 μmol/m2/s (white fluorescent light was continuously and uniformly irradiated)
- Sterile test conditions: yes/no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: yes
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
82 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL (71 - 98 mg/L)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Dunnett multiple comparison method
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of influence to the test organism: For all tested groups mutation of cell or emersion of abnormal cell were not observed.
- Observation of condition of test solution: At the beginning of the exposure, it was confirmed by visual observation that the test solutions for all of the concentration groups were clear and colorless. Further, at the end of exposure, there was no change in condition of the test solution in comparison with state of the beginning of the exposure.
- Concentration of the test substance in the test solution: For all concentration groups, concentrations of the test substance in the test solution were analyzed at the beginning of the exposure and at the end of the exposure. Ratios of measured concentrations in each concentration group to the initial concentration were 68 - 92 % at the end of the exposure. Further average concentrations calculated by geometric average in each concentration groups were 3.4 mg/l for 4.6 mg/l group, 8.5 mg/l for 10 mg/l group, 19.6 mg/l for 22 mg/l group, 44.7 mg/l for 46 mg/l group and 88.1 mg/l for 100 mg/l group.
- Cell concentration and average value: Average cell concentrations after 72 hours were 181.63 x 10^4 cells/mL in 3.4 mg/L group, 157.53 x 10^4 cells/ml in 8.5 mg/L group, 120.16 x 10^4 cells/mL in 19.6 mg/L group, 60.99 x 10^4 cells/mL in 44.7 mg/L group and 9.87 x 10^4 cells/mL in 88.1 mg/L group. Average cell concentration was 160.38 x 10^4 cells/mL in control group.
- Validity of the test: Average cell concentration of the control group at the end of exposure period was 160.38 x 104 cells/ml, fluctuation coefficient of daily growth rate during exposure period was 1.3 % in the control group and fluctuation coefficient of growth rate in replicates of the control group was 0.8 %. Since all these data are sufficient for fulfillment of conditions of the Growth Inhibition Test, i.e. cell concentration of the control group should be 16-fold growth or more at the end of exposure period in comparison with the start of exposure period, coefficient of daily growth rate during exposure period of the control group should be 35 % or less and fluctuation coefficient of growth rate in replicates of the control group should be 7 % or less, the validity of the present test was confirmed.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 0.85 mg/L
Reported statistics and error estimates:
- EC50 was determined by using Logit method from inhibition of growth rate (%) calculated by the Rate-based method. Note that EC50 calculated by the Rate-based method is expressed as ErC50(0 - 72 hr).
- NOEC was obtained as the maximum concentration which has no significant difference with the control group by using Dunnett multiple comparison method (One side, significance level: α = 0.05). Note that NOEC derived by the rate-based method is express as NOEC (Rate-based method: 0 - 72hr).
- For calculation of results, statistical software "StatLight 2000 (Yukms Co.Ltd.)" was used.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study it was found that HBPA has a ErC50 82 mg/l.

Description of key information

After 72 hours exposure test

ErC50: 82 mg/L (71 - 98 mg/L for 95 % confidence limits)

NOEC: 8.5 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
82 mg/L
EC10 or NOEC for freshwater algae:
8.5 mg/L

Additional information

An algal growth inhibition study was conducted (Ministry of the Environment Government of Japan, 2007, Study 18011) to determine the effect of HBPA on the growth of Pseudokirchneriella subcapitata. The study was conducted according to Heisei 15/11/21-No.1·121002 of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Heisei 15/11/13-No.2 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry and No.031121002 of the Environmental Policy Bureau, Ministry of the Environment, Japan, and in compliance with GLP.

Triplicate algal cultures were exposed to aqueous solutions of the test item at nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L (calculated geometric average 3.4, 8.5, 19.6, 44.7 and 88.1 mg/L) for 72 hours under static test conditions and constant illumination. Six replicate control cultures were also incubated concurrently with the test samples.

Analysis of the fresh and old test preparations showed the measured concentrations were outside the 68 – 92 % range; hence, the results are expressed in terms of geometric measured concentrations.

The growth rate of Pseudokirchneriella subcapitata was affected by the presence of HBPA at the test concentrations. The EC50 and NOEC for growth rate were 82 and 8.5 mg/L, respectively.