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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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To achieve the nominal test concentrations 1.00, 2.15, 4.64, 10.00, 21.50 and 46.40 g of test substance were added to the test water using an ultra-turrax stirrer, respectively. A total of 25 minutes were needed from the beginning of test solution preparations until their termination. Once all solutions were ready the fish were subsequently placed then placed into the aquaria. (For all concentrations the fish were placed in the unfiltered test solution, the test solution was only filtered before analysis)
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Length at study initiation (length definition, mean, range and SD): 3.25 cm (2.9 - 3.8 cm)
- Weight at study initiation (mean and range, SD): 0.21 g (0.13 - 0.37 g)
- Corpulence factor of the batch: 0.6
- Feeding during test: Withdrawal of food: 1 day before and during exposure
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Nominal and measured concentrations:
Nominal test concentrations (mg/L):
0, 100, 215, 464, 1000, 2150, 4640
Details on test conditions:
TEST SYSTEM
- Fill volume: 10 L
- Aeration: None
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.2 g fish / L water

OTHER TEST CONDITIONS
- Photoperiod: Photoperiod: 16 : 8 hours day-night regime

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Results used to determine the conditions for the definitive study: LC50 after 96 h: between 1000 and 10000 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
271.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
other: LC20
Effect conc.:
604.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1 216.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
1 422.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Reported statistics and error estimates:
The LD50 was statistically evaluated from the LC20 value (604.5 mg/L) and the LD100 value (1422.3 mg/L).

LC50 (1 h): > 4640* mg/L

LC50 (4 h): ca. 4640* mg/L

LC50 (24 h): 3474 (2476 - 5906)* mg/L

LC50 (48, 72, 96 h): > 2150 < 4640* mg/L

2 and 10 fish exposed to 2150* and 4640* mg test substance/L died  respectively. No deaths were observed at 1000* mg/L and less. Symptoms of  toxicity (gasping and slight apathy) were limited to the highest dose  groups.

(* = nominal concentration)

LC50 after 1 and 96 hours

(Based on the mean analytically detected concentrations, unfiltered  samples)

Analytical / No of  /   dead     /         /

conc.      / fish   /   fish     /  mort.  /

(mg/l)     / Start  / after 96 h /    %    /

--------------------------------------------

16.8       /  10    /   0        /  0.0    /

33.8       /  10    /   0        /  0.0    /

101.5      /  10    /   0        /  0.0    /

271.3      /  10    /   0        /  0.0    /

604.5      /  10    /   2        / 20.0 *  /

1422.3     /  10    /  10        / 100.0 * /

LC50 > 604.5 (10 % significance level)

LC50 < 1422.3 (1 % significance level)

ANALYTICAL MONITORING:

The analytically detected concentrations of the unfiltered samples were  initially in the range of 29.5 % - 59.5 % and after about 96 h between  1.2 - 4.4 % of the nominal concentrations.

The analytically detected concentrations of the filtered samples were  initially in the range of 0.6 - 2.0 % and after about 96 hours between  0.0 - 1.0 % of the nominal concentrations.

OBSERVATIONS:

All test solutions showed a certain level of turbidity (milky in colour)  and fat-like drops were visible on the water surface, both increasing  with each increase in the concentration of the test substance  respectively.

Due to the high level of turbidity within the two highest concentrations  (2150 and 4640 mg/L) it was necessary to direct the fish the front pane  of the aquaria using a net so that observations for fish mortalities or  for possible symptoms of toxicity could be made.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10/01/2000-18/05/2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study according to international guideline (OECD Guideline 203) under GLP conditions, validity criteria met.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water accomodated fractions (WAF) were prepared at 10 and 100 mg/l and dilutions containing 10 % and 1 % of the 10 mg/L WAF (1.0 and 0.1 mg/L).
- Sampling method: During the combined limit/range-finding test amples were taken from all test solutions including the blank-control for possible analysis. Samples were taken after 0, 24 and 96 hours. 10 mL from the approximate center of the vessels was sampled.
- Sample storage conditions before analysis: The samples were stored in a freezer until analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accomodated fractions (WAFs) were prepared at 10 and 100 mg 2-Ethylhexyl 4-methoxycinnamate per litre, mixed with test medium and stirred for ca. 48 hours in a closed vessel in the dark. Then, the dispersions were allowed to settle overnight until a stable separation of liquid phases was reached. Subsequently, the water phase was decanted from the test substance phase and used for testing. A test substance film was still observed at the water phase obtained from the WAF at 100 mg/L separation. Therefore, this solution was put into a clean separating funnel and allowed to stabilise for an additional hour. After separation, the water phase of the solution was decanted through glass wool. the test concentrations of 0.1 and 1.0 mg/l were prepared by dilution of the WAF prepared at 10 mg/L.
- Controls: Yes, test medium without test substance or additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No, all final test solutions were clear and colourless without precipitation or a test substance film.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Source: Zodiac, proefacc. "De Haar Vissen", Wageningen, the Neteherlands
- Length at study initiation (length definition, mean, range and SD): 2 ± 0.2 cm
- Weight at study initiation (mean and range, SD): 0.27 ± 0.05 g
- Method of breeding: No data, F1 offspring from a single parent=pair bred in UV-tretaed water
- Feeding during test: No feeding during test

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Acclimation conditions: Same as test
- Type and amount of food: Trouvit
- Feeding frequency: Daily
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
Combined range-finding/limit test
Hardness:
250 mg/L CaCO3
Test temperature:
19.5 - 20.8 °C
pH:
7.5 - 8.0
Dissolved oxygen:
6.2 - 9.9 mg/L
Salinity:
Not relevant
Nominal and measured concentrations:
Nominal: 0.1, 1.0, 10 and 100 mg/L
Measured: only measured for 100 mg/L nominal: 0.71 mg/L (t=0), 0.36 mg/L (t=24 h), 0.075 mg/L (t=96 h)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: glass, 5 litre
- Aeration: Aeration was introduced after 72 hours of exposure
- No. of organisms per vessel: 7 for control and 100 mg/L, 3 for 0.1, 1.0 and 10 mg/L
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: Control: 7 fish per 3 litres of medium, test concentrations: 3 fish per 2 litres of medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Ro water (tapwater purified by reverse osmosis)
- Metals: Mg2+: 12 mg/L; Na+: 15 mg/L; K+: 3 mg/L
- Chlorine: Cl-: 145 mg/L
- Ca/mg ratio: 6.7
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 light/dark

EFFECT PARAMETERS MEASURED: Mortality

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: Not relevant, limit test
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L (nominal)
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: None reported
- Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: results indicate that 2-Ethylhexyl 4-methoxycinnamate concentrations at the solubility level are not stable and decrease exponentially at 0.67^-0.023t.
- Effect concentrations exceeding solubility of substance in test medium: Yes, owing to the limited solubility of 2-Ethylhexyl 4-methoxycinnamate in water (0.22 - 0.75 mg/L), concentration levels toxic for carp could not be reached in WAFs prepared at loadings up to 100 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality: 100 % at highest concentrations
- LC50: 96h LC50: 0.14 mg/L
Reported statistics and error estimates:
No LC50 could be calculated because the test substance proved to be non-toxic (LC50 > maximum concentration).
Validity criteria fulfilled:
yes
Conclusions:
Toxicity of 2-Ethylhexyl 4-methoxycinnamate towards Cyprinus carpio was investigated. Owing to the limited solubility of 2-Ethylhexyl 4-methoxycinnamate in water (0.22 - 0.75 mg/L), concentration levels toxic for carp could not be reached in WAFs prepared at loadings up to 100 mg/L.
Executive summary:

Toxicity of 2-Ethylhexyl 4-methoxycinnamate towards Cyprinus carpio was investigated according to OECD Guideline 203 under GLP conditions. The 96h LC50 was found to be > 100 mg/L. Owing to the limited solubility of 2-Ethylhexyl 4-methoxycinnamate in water (0.22 - 0.75 mg/L), concentration levels toxic for carp could not be reached in WAFs prepared at loadings up to 100 mg/L. Validity criteria were met, although some minor deviations from these citeria were observed.

Description of key information

Short-term toxicity to fish: > 100 mg/L (96h-LC50)

Key value for chemical safety assessment

Additional information

The toxicity of the test substance towards fish was tested in a Water Accommodated Fraction (WAF) with loading rates up to 100 mg/L and Cyprinus carpio. Actual test concentrations at 100 mg/L loading were below 1 mg/L. The 96h-LC50 was > 100 mg/L loading (OECD guideline 203, GLP, reliability 1). This study was selected as the key study. In a supporting study with Danio rerio (OECD 203, GLP, reliability 1) a (milky) solution was prepared by stirring. The 96h-LC50 was reported as 1216.1 mg/L whereas the 96h-NOEC was 271 mg/L.