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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
Study period:
2009-09-28 - 2009-10-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: ‘Testing Methods for New Chemical Substances’ of Pharmaceutical and Food Safety Bureau (PFSB), Ministry of Health, Labour and Welfare
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Environmental Policy Bureau-Ministry of the Environment (November 21, 2003 No. 1121002; November 13, 2003, No. 2; Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry No. 031121002)
Deviations:
not specified
GLP compliance:
yes
Remarks:
"Standard Concerning Testing Facility Relating to New Chemical Substances" (November 21, 2003 No. 1121003; PFSB, Ministry of Health, Labour and Welfare November 17, 2003, No. 3; Environmental Policy Bureau, Ministry of the Environment No. 031121004)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.250, 0.430, 0.720, 1.23 and 2.09 mg/L and control
- Sampling interval: Test substance concentration was analysed every 24h.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Defined amounts of the test material were disolved in test medium (OECD) as homogeneously as possible, to obtain the test solutions of the desired test concentrations.
- Controls: OECD test medium only
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection
- Method of cultivation: Aseptically subcultured using the plant culture medium ATCC Culture Medium 625.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.9 - 23.4 °C (in the incubation chamber)
Temperature in the incubators were measured every 24 hours from the start of exposure to the end of exposure.
pH:
7.7 - 7.9 at test start
7.8 - 8.5 at test end (after 72 h)
pH value of the test solution was measured for all test groups at the beginning of exposure and at the end of exposure. At start of exposure, measurement was performed by using the remainder of the prepared test solution. At the end of the exposure, the pH value of the test solution in one test container was measured in each test group.
Nominal and measured concentrations:
Nominal: 0.250, 0.430, 0.720, 1.23 and 2.09 mg/L (spacing factor = 1.7) and control
Initial measured concentrations (0h): 0.202, 0.371, 0.639, 1.08 and 1.87 mg/L
Mean measured concetrations (24h): 0.167, 0.283, 0.479, 0.852 and 1.42 mg/L
Mean measured concentrations (48h): 0.126, 0.284, 0.441, 0.785 and 1.37 mg/L
Mean measured concentrations (72h): 0.016, 0.153, 0.361, 0.678 and 1.27 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300mL glass triangle flask (breathable silicone stopper)
- Type (delete if not applicable): open system with shaking (100 rpm) in an incubator
- Initial cells density: 10,000 cells/mL.
- Control end cells density: 1,381,687 cells/mL
- No. of vessels per concentration (replicates): 3/exposure level
- No. of vessels per control (replicates): 6/control level

GROWTH MEDIUM
- Standard medium used: Yes, OECD medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD test medium (see "Any other information on materials and methods incl. tables")
- Culture medium different from test medium: Yes. OECD medium was used as test medium and ATCC Culture medium 625 was used as culture medium.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24h light
- Light condition: Continuous illumination using a fluorescent light (wavelength: 400~ 700 nm, light quantum: 66.19 - 67.50 μmol·m-2·s-1).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Beckman Coulter Cell Counting Analyzer (Model: Z2))

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Test concentrations: 0.250, 0.430, 0.720, 1.23 and 2.09 mg/L and control
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.553 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95%-confidence limits: 0.480 - 0.636 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.202 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Cell morphology of all the test groups at the end of the exposure, no abnormality such as change in shape or agglomeration was observed.
- Any stimulation of growth found in any treatment: A slight stimulation of growth was determined at the lowest test concentration of 0.202 mg/L (initial measured). As this stimulation was only 0.3 % (average value of 3 replicates) it was not significant compared to the control. Accordingly, it was considered to be a consequence of biological variation of algal growth and it was not regarded to be treatment related.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Stability under exposure condition was carried out for nominal concentration 0.250 mg/L and 16.0 mg/L. The initial test concentration was 0.225 mg/L and 16.2 mg/L. 72hr later, the concentration was 0.174 mg/L and 12.3 mg/L. At the same time, 72hr later the concentration was 0.239 mg/L and 15.5 mg/L if protection from light was applied. Therefore it was judged the substance is unstable to light.
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- EC50 (72h): 1.21 mg/L for growth rate
- Other: An algae growth inhibition test using the reference substance potassium dichromate was conducted with the test organism to confirm the reproducibility (2009-06-29 - 2009-07-02). The ErC50(0~72hr) was determined to be 1.21mg/L, within the range of the lab (average ±30%: 0.65 ~ 1.22mg/L, N=7, from May 2005).
Validity criteria fulfilled:
yes
Conclusions:
The study was performed under GLP according to relevant Japanese test guidelines, without deviations which may have impact on the validity of the study. Thus, the results were obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable. According to the reported 72h-EC50 of 0.553 mg/L for the parameter growth rate, the test substance possesses a certain toxic potential towards aquatic algae.
Executive summary:

In the present study the acute toxicity of methyl styryl keton (Benzalaceton) towards aquatic algae was investigated according to relevant Japanese test guidelines (‘Testing Methods for New Chemical Substances’ of Pharmaceutical and Food Safety Bureau (PFSB), Ministry of Health, Labour and Welfare, as well as Environmental Policy Bureau-Ministry of the Environment (November 21, 2003 No. 1121002; November 13, 2003, No. 2; Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry No. 031121002)), without deviations which may have impact on the validity of the study. No GLP certificate is included, however, the study was performed under GLP compliance.

The freshwater green algal species Pseudokirchneriella subcapitata was used as test organism and exposed to test substance concentrations of 0.250, 0.430, 0.720, 1.23 and 2.09 mg/L for 72 hours. A control was included and the reference substance potassium dichromate was tested at regular intervals. Under the conditions used for the test, the following effect concentrations were determined based on the inhibition of the parameter growth rate compared to control cultures:

72h-ErC50 = 0.553 mg/L

72h-NOErC = 0.202 mg/L

As a conclusion of the analytical part of this study, it can be stated that the desrease in the concentration of the test substance in test solutions could be considered to be the reason of photodegradation and adsorption to algal bodies as main cause. Within a parallel light irridation study, photodecomposability was confirmed, as the ratio of the measured concentration at the end of exposure to the preset concentration in the light irradiation study area (setting concentration: 0.250 mg/L, cell-free inoculation) is 65 % under the light condition and 82 % at the dark condition. The measured concentration in the test solutions is 81 - 89 % of nominal concentration at test start and 6 - 61 % of nominal concentration at test end. As indicated above, the main reason for the concentration decrease is thought to be due to adsorption by algae. The biological results were based on initial mean measured concentrations of the test substance accordingly.

Description of key information

Japanese guideline study (GLP), Pseudokirchneriella subcapitata: EC50 (72h) growth rate = 0.553 mg/L (initial mean), NOECr (72h) = 0.202 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
0.553 mg/L

Additional information

The acute toxicity of methyl styryl keton (Benzalaceton) towards aquatic algae was investigated according to relevant Japanese test guidelines (‘Testing Methods for New Chemical Substances’ of Pharmaceutical and Food Safety Bureau (PFSB), Ministry of Health, Labour and Welfare, as well as Environmental Policy Bureau-Ministry of the Environment (November 21, 2003 No. 1121002; November 13, 2003, No. 2; Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry No. 031121002)), without deviations which may have impact on the validity of the study. No GLP certificate is included, however, the study was performed under GLP compliance. The freshwater green algal species Pseudokirchneriella subcapitata was used as test organism and exposed to test substance concentrations of 0.250, 0.430, 0.720, 1.23 and 2.09 mg/L for 72 hours. A control was included and the reference substance potassium dichromate was tested at regular intervals. Under the conditions used for the test a 72h-EC50 of 0.553 mg/L and a 72h-NOEC of 0.202 mg/L was determined based on the inhibition of the parameter growth rate compared to control cultures. As a conclusion of the analytical part of this study, it can be stated that the decrease in the concentration of the test substance in test solutions could be considered to be the reason of photodegradation and adsorption to algal bodies as main cause. Within a parallel light irridation study, photodecomposability was confirmed, as the ratio of the measured concentration at the end of exposure to the preset concentration in the light irradiation study area (setting concentration: 0.250 mg/L, cell-free inoculation) is 65 % under the light condition and 82 % at the dark condition. The measured concentration in the test solutions is 81 - 89 % of nominal at test start and 6 - 61 % of nominal at test end. As indicated above, the main reason for the concentration decrease is thought to be due to adsorption by algae. The biological results were based on initial mean measured concentrations of the test substance accordingly.

As supporting information, the acute toxicity to algae of the test substance was predicted by the computer program ECOSAR v1.11 (EPIWIN v4.1) by US EPA. An extensive set of structure – activity relationships (SARs) is utilized by the computer program, to predict the acute and chronic toxicity for three trophic levels for aquatic species.The mode of toxic action for most neutral organic chemicals is narcosis, and many chemical classes present toxicity to organisms via narcosis (i.e. ethers, alcohols, ketones). However, some organic chemical classes have been identified as having a more specific mode of toxicity. These are typically organics that are reactive and ionizable and that exhibit excess toxicity in addition to narcosis (i.e. acrylates, epoxides, anilines).The query substance belongs to the specific EOSAR class of Vinyl/Allyl Ketones. However, the SAR for neutral organics is more reliable due to the larger training set. In addition, the result with the neutral organic SAR is lower than with the specific Vinyl/Allyl Ketones SAR. This indicates that narcosis induced by the neutral organic is the primary toxic effect of benzalacetone. For algae an EC50 value of 47.848 mg/L is predicted with the neutral organic SAR.

Regarding long-term toxicity, the so-called Chronic Value (ChV) is predicted, which is defined as the geometric mean of the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC). For the test substance a ChV(4d) of 12.627 mg/L is predicted, indicating a moderate toxicity concern.