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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-07-03 - 2006-07-07
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: 1.0, 1.8, 3.2, 5.6, 10 and 18 mg/L and a control
- Sampling method: For analytical monitoring samples of the freshly prepared test solutions were taken at test start and at test medium renewal after 72 hours. In addition, samples of the aged test solutions (24 hours old) were taken after 24 and 96 hours. The samples were collected from the middle layer of each test container and analyzed by HPLC method.
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 water (dechlorinated tap water) as homogeneously as possible, to obtain the test solutions of the desired test concentrations
- Controls: Test medium only .
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No. Visual check and filtration by 22 μm membrane filter confirmed > 400 mg/L solubility by measuring the concentration of the test substance by HPLC.
Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Common name: Himedaka (Oryzias latipes)
- Source: in-house reproduction
- Length at study initiation (length definition, mean, range and SD): 2.3 cm (2.1 - 2.6 cm), n=10
- Weight at study initiation (mean and range, SD): 0.10 g (0.06 - 0.16 g), n=10

ACCLIMATION
- Acclimation period: 2006-03-13 - 2006-07-03 (12 month aged)
Water: same water as used for the test
Method: flow-through
Temperature: 24 ± 1 °C
Light: Indoor light, 16-hour light/8-hour dark
- Type and amount of food during acclimation: Tetramine (quantity: about 2% body weight/day)
- Feeding frequency during acclimation: Feeding was stopped 24 hours before exposure (test start).
- Health during acclimation (any mortality observed): During acclimation period, the successive 7-day mortality rate was 0.6%, healthy and normal individuals by visual check were used for the test.

FEEDING DURING TEST
- Frequency: No feeding during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
freshly prepared test solutions: 24.0 °C
aged test solutions (after 24 h exposure): 24.0 °C
pH:
freshly prepared test solutions: 7.8
aged test solutions (after 24 h exposure): 7.3 - 7.7
Dissolved oxygen:
freshly prepared test solutions: 8.2 mg/L
aged test solutions (after 24 h exposure): 7.0 - 7.9 mg/L
Nominal and measured concentrations:
Nominal concentrations: 1.0, 1.8, 3.2, 5.6, 10 and 18 mg/L (spacing factor 1.8) and a control
Time-weighted mean measured concentrations: 0.744, 1.42, 2.69, 4.89, 8.92 and 16.5 mg/L and a control
Details on test conditions:
TEST SYSTEM
- Test vessel: container
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 5 L/ test vessel
- Renewal rate of test solution (frequency/flow rate): The test was conducted semi-static with a water renewal every 24 hours.
- No. of organisms per vessel: 10 fish/vessel
- No. of vessels per concentration (replicates): 1 vessel/test group
- No. of vessels per control (replicates): 1 vessel/control group

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated water (tap water after treated by activated carbon and then aerated, see "Any other information on materials and methods incl. tables")
- Culture medium different from test medium: no
- Intervals of water quality measurement: measurement of test water used for the test: 2006-06-21.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light / 8-hour dark
- Light intensity: Indoor light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : At exposure beginning, as well as 3, 6, 24, 48, 72 and 96 hours after exposure the number of dead fish was recorded. In addition the observed signs of toxicity and any abnormalities were recorded. Dead fish were promptly removed not to deteriorate the water quality.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study : According to preliminary test results the 96h-LC0 was determined to be 1 mg/L and the 96h-LC100 was determined to be 10 mg/L. Based on these results the concentrations for the main experiment were selected.
- Test concentrations: 1.0, 1.8, 3.2, 5.6, 10 and 18 mg/L and control
Reference substance (positive control):
not required
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
11 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95%-confidence limits: 9.1 - 21 mg/L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
7.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
95%-confidence limits: 5.6 - 10 mg/L
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
5.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95%-confidence limits: 4.7 - 13 mg/L
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
5.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95%-confidence limits: 4.7 - 13 mg/L
Details on results:
- Other biological observations: Environmental factors that seemed to have influenced the reliability of the test results were not recognized.
- Mortality of control: No mortality was observed in the control group.
- Other adverse effects control: No sublethal effects were observed in the control group.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Although concentration change of the test substance could be considered partly due to analytical error, the effect of volatilization and photodegradation was judged to be the main cause.
- Effect concentrations exceeding solubility of substance in test medium: No. Visual check and filtration by 22 μm membrane filter confirmed >400 mg/L solubility by measuring the concentration of the test substance by HPLC.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
LC50`s were calculated by the Logit method.
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 96h-LC50 value of 5.7 mg/L the test substance possesses a certain toxic potential towards fish.
Executive summary:

In the present study the acute toxicity of Benzalaceton towards fish 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.

Himedaka (Oryzias latipes) was used as test organisms. 10 animals per test group were exposed to test substance concentrations of 1.0, 1.8, 3.2, 5.6, 10 and 18 mg/L for 96 hours in a semi-static test. A control was included, no reference substance is mentioned. Under the conditions used for the test, the following effect concentrations were determined based on mortality and other sublethal effects observed:

96h-LC5 0= 5.7 mg/L

96h-LC0 = 2.7 mg/L

96h-LC100 = 8.9 mg/L

96h-NOEC = 2.7 mg/L

As a conclusion of the analytical part of this study, it can be stated that the concentration change of the test substance could be considered partly due to analytical error, but the effect of volatilization and photodegradation was judged to be the main cause. In the freshly prepared test solutions recoveries ranged from 95 - 99 %, while recoveries were reduced to 50 - 92 % in the aged test solutions (24 h old). Therefore, the time weighted average value of each measured value (when exposure started, before water replacement at 24 hours, after water replacement at 72 hours, when exposure ended, the logarithmic average values were calculated, and the arithmetic average value thereof was calculated. The biological results were based on the time-weighted mean measured concentrations of the test substance accordingly.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-01-11 - 1993-01-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes
Remarks:
No certificate included.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:
No surrogate or analogue material was used.
Analytical monitoring:
yes
Details on sampling:
Samples were taken each time after preparation of the test concentrations and at renewal (24 h later).
Vehicle:
no
Details on test solutions:
The substance was applied by direct weight and was grinded with a mortar and treated for 60 sec with 8000 rpm in an Ultra-Turrax before.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Strain: Hamilton Buchanan
- Source: West-Aquarium (Bad Lauterberg)
- Date of birth: 1992-05-18
- Date of delivery: 1992-08-11
- Length at study initiation: 2.5 - 3.5 cm

ACCLIMATION
- Medical pretreatment: none
- Health during acclimation: mortality < 2 %
- Water used: synthetic freshwater according to ISO
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No post exposure observation period described.
Hardness:
13.3 °dH
Test temperature:
Control: 20.6 - 22.3 °C
5.5 mg/L and 7.8 mg/L: 20.6 - 22.8 °C
11 mg/L: 20.5 - 22.8 mg/L
16 mg/L: 21.0 - 22.7 mg/L
22 mg/L: 21.4 - 22.8 mg/L
pH:
Control: 7.6 - 8.1
5.5 mg/L: 7.6 - 8.2
7.8 mg/L: 7.7 - 8.2
11 mg/L: 7.7 - 8.3
16 mg/L: 7.4 - 8.3
22 mg/L: 7.7 - 8.3
Dissolved oxygen:
Control: 8.0 - 10.8 mgO2/L (Saturation: 93.8 - 123.4 %)
5.5 mg/L: 8.2 - 11.4 mgO2/L (Saturation: 96.9 - 130.3 %)
7.8 mg/L: 8.4 - 11.0 mgO2/L (Saturation: 97.9 - 125.7 %)
11 mg/L: 8.4 - 11.7 mgO2/L (Saturation: 97.8 - 133.6 %)
16 mg/L: 8.5 - 9.6 mgO2/L (Saturation: 98.7 - 103.7 %)
22 mg/L: 8.5 - 8.6 mgO2/L (Saturation: 98.6 - 102.3 %)
Salinity:
Not applicable.
Nominal and measured concentrations:
5.5, 7.8, 11, 16 and 22 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: fish tanks, 300 x 135 x 200 mm
- Medium: 5 L
- Aeration: yes
- Renewal rate of test solution: daily

BIOMASS LOADING RATE
- Control: 2.93 g
- 5.5 mg/L: 3.08 g
- 7.8 mg/L: 2.89 g
- 11 mg/L: 3.93 g
- 16 mg/L: 4.66 g
- 22 mg/L: 3.87 g

TEST MEDIUM / WATER: synthetic freshwater according to ISO

OTHER TEST CONDITIONS
- Photoperiod: 8 h light, 16 h dark
- Application of test substance: direct weight
- Adjuvant: Ultra-Turrax
Reference substance (positive control):
not required
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
5.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
6.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
8.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
7.8 mg/L: 1 animal with abnormal swimposition and lethargic swim behaviour after 72 h; 8 animals with lethargic swim behaviour as well as 2 animals with additional abnormal swim position after 96 h
11 mg/L: 10 animals with lethargic swim behaviour after 24 h; 5 animals with lethargic swim behaviour and 5 animals with uncontrolled behaviour and abnormal swim position after 48 h. After 72 h 1 animal was motionless, uncontrolled swim behaviour after contact
16 mg/L: 9 animals with abnormal swim position and lethargic swim behaviour
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
The arithmetic mean of the analytically determined values was used to report the LC0 and LC100. The LC50 was determined as geometrical mean of these results.
Validity criteria fulfilled:
yes
Remarks:
Basic information available.
Conclusions:
The study is regarded as a valid guideline study with GLP compliance. According to the reported LC50 value (6.5 mg/L) the test substance possesses a certain toxic potential towards fish.
Executive summary:

The acute toxicity of the substance 4 -phenylbutenone was investigeted according to EU Method C.1 (which is in most parts equivalent to OECD Guideline 203) (Kanne, 1993). No GLP certificate is included, however, the study was performed under GLP compliance. Danio rerio (Zebra fish) was used as test organisms and where exposed for 96 hours to the test substance. A control was included, no reference substance is mentioned. It was a semi-static freshwater test with substance concentrations of 5.5, 7.8, 11, 16 and 22 mg/L. The substance was applied by direct weight and was grinded with a mortar and treated for 60 sec with 8000 rpm in an Ultra-Turrax before. A 8 hours light / 16 h dark cycle was applied. As results a LC0 of 5.1 mg/L and a LC100 of 8.3 mg/L were reported, using the arithmetic mean of the analytically determined concentrations. The LC50 of 6.5 mg/L was determined by the geometric mean of the obtained values mentioned before. All values were reported after the exposition period of 96 hours.

Description of key information

Japanese guideline study (GLP),Oryzias latipes (Himedaka): LC50 (96h) = 5.7 mg/L (time-weighted mean)

EU Method C.1 (GLP), Danio rerio (Zebra fish): LC50 (96h) = 6.5 mg/L (geometric mean)

Key value for chemical safety assessment

LC50 for freshwater fish:
5.7 mg/L

Additional information

The acute toxicity of Benzalaceton towards fish 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 (Kureha, 2006). No GLP certificate is included, however, the study was performed under GLP compliance. Himedaka (Oryzias latipes) was used as test organisms. 10 animals per test group were exposed to test substance concentrations of 1.0, 1.8, 3.2, 5.6, 10 and 18 mg/L for 96 hours in a semi-static test. A control was included, no reference substance is mentioned. Under the conditions used for the test a 96h-LC0 of 2.7 mg/L, a 96h-LC50 of 5.7 and a 96h-LC100 of 8.9 mg/L were determined based on mortality and other sublethal effects observed. As a conclusion of the analytical part of this study, it can be stated that the concentration change of the test substance could be considered partly due to analytical error, but the effect of volatilization and photodegradation was judged to be the main cause. In the freshly prepared test solutions recoveries ranged from 95 - 99 %, while recoveries were reduced to 50 - 92 % in the aged test solutions (24 h old). Therefore, the time weighted average value of each measured value (when exposure started, before water replacement at 24 hours, after water replacement at 72 hours, when exposure ended, the logarithmic average values were calculated, and the arithmetic average value thereof was calculated) was calculated. The biological results were based on the time-weighted mean measured concentrations of the test substance accordingly.

In the study from Kanne (1993), the acute toxicity of the substance 4-phenylbutenone was investigated according to EU Method C.1 (which is equivalent to OECD Guideline 203). No GLP certificate is included, however, the study was performed under GLP compliance. Danio rerio (Zebra fish) was used as test organisms and was exposed for 96 hours to the test substance. A control was included, no reference substance is mentioned. It was a semi-static freshwater test with substance concentrations of 5.5, 7.8, 11, 16 and 22 mg/L. The substance was applied by direct weight and was grinded with a mortar and treated for 60 sec with 8000 rpm in an Ultra-Turrax before. An 8 hours light / 16 h dark cycle was applied. As results a LC0 of 5.1 mg/L and a LC100 of 8.3 mg/L were reported, using the arithmetic mean of the analytically determined concentrations. The LC50 of 6.5 mg/L was determined by the geometric mean of the obtained values mentioned before. All values are based on an exposure period of 96 hours.

Supporting information is given by the prediction with the computer program ECOSAR v1.11 (EPIWIN software) 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 fish an LC50 (96 h) of 111.132 mg/L is predicted with the neutral organic SAR.

Brorson et al. (1994) investigated the potential environmental impact of wastewater from a chemical-pharmaceutical plant. For this purpose, the test substance was investigated in a mixture for its toxicity potential towards the freshwater fish Brachydanio rerio (new name: Danio rerio). The experimental part was conducted as semi-static test with an exposure duration of 96 h. It was part of the STORK project of the Swedish Environmental Protection Agency. The complex effluent of a wastewater mixture which is dominated by the activities of a chemical plant, including benzylidene acetone (CAS 122-57-6) was used for testing. Degraded as well as nondegraded test sample were used and none of them showed any lethal effect to zebra fish. However, at test concentrations > 60 %, normal swimming of fish was disturbed, making them rest at the bottom of the vessel. Gill movements were not affected and all fishes were capable of swimming. The LC50(96h) value is therefore reported to be > 0.21 %, corresponding to a value of 2100 mg/L.