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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-01-15 to 2002-01-17
Reliability:
2 (reliable with restrictions)
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples totalling 5 mL were taken at T24 and T48 hours from the LS test vessels containing daphnids and stored at -20°C until analysis. Samples were thawed at room temperature. An aliquot of each sample was placed in a tube and diluted ten-fold in ethanol. If the sample concentration was higher than 0.5 mg/L then another dilution in ethanol was made to achieve concentrations in the range from 0.01 to 0.5 mg/L of test item.
Vehicle:
no
Details on test solutions:
The stock solution, for the limit and range-finding tests, was prepared by dispersing the test item directly in M4 reconstituted water.
The condition of preparation of the stock solution are reported as follows:
Quantity of test item: 100 mg
Volume of M4 reconstituted water: 1000 mL
Concentration of the test item: 100 mg/L
Duration of the agitation: 23h 50min

A loading rate of 100 mg/L was used to prepare the stock solution. As test item droplets were still in suspension after agitation, the stock solution was filtered through a filter of porosity 0.45 µm to remove the suspended fraction. The filtered solution, corresponding to the limit of solubility of the test item under our experimental conditions (LS), was then used to prepare the test solutions.
Test solutions were prepared by further dilution of the LS solution with M4 reconstituted water to provide a geometric series of concentrations: 0 mg/L, LS/1000, LS/100, LS/10 and LS.
Test vessels containing daphnids were filled directly from the test solution containers immediately after preparation and test solutions remained unchanged.
The pH of the test solutions remained within acceptable limits (between 6.5 and 9 and in the range ± 0.5 unit of the test water) after preparation and there was no adjustment of pH before addition of the animals.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna STRAUS-clone 5
- Source: Centre Technique du Bois et de l'Ameublement, 75012 Paris, France
- Age at study initiation (mean and range, SD): at the beginning of the test, first instar daphnids between 6 and 24 hours old and produced from pathogenically reproducing brood female population
- Feeding during test
Culture animals are fed with a diet of between 0.1 and 0.2 mg of carbon per Daphnia per day, in the form of the algal strain Scenedesmus subspicatus which is cultures at CIT. Animals were not fed during the test.

ACCLIMATION
Animals are held in water of the same quality at the test water from birth until use in the test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
CaCO3: 306 to 323 mg/L
Test temperature:
19.4 to 20°C
pH:
pH: 7.80 to 8.34
Dissolved oxygen:
8.0 to 8.7 mg/L (> 60 % of the air saturation value)
Nominal and measured concentrations:
nominal concentrations: 0 mg/L, LS/1000, LS/100, LS/10 and LS.
LS: limit of solubility obtained from an initial loading rate of 100 mg/L
Details on test conditions:
TEST SYSTEM
Environmental conditions during culture
Water: reconstituted (M4). The pH of this solution is 8.0 ± 0.5; the sum of Ca and Mg ions in the solution is 2.5 mmol/L; total hardness is generally between 238 and 289 mg/L as CaCO3
Temperature in water: between 18 °C and 22 °C. Temperature is recorded continuously and checked generally two times a week
Light/dark cycle: 16/8 hours
Forced aeration: none. Oxygen concentrations remain above 80% of saturation value at the selected temperature
Loading: the animals are loaded at one animal/20 mL of culture water

Environmental conditions during the test
Water: see above
Temperature in water: between 18 °C and 22 °C and not varying by more than ± 1°C throughout the test, measured using a max-min thermometer
Light/dark cycle: 16/8 hours
Duration of test: 48 hours
Loading: test vessels were loaded at one daphnia for 10 mL of solution
pH: between 6.5 and 9 and did not vary by more than 1 unit during the test
Forced aeration: was not used during the test. Dilution water was aerated prior to the addition of the test item
Oxygen concentrations: were maintained at > 60% of the air saturation value at that temperature
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.518 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No immobilization of Daphnia was observed at 0, 0.1, 1.0, 10 and 100 mg/L throughout the duration of the test. the test item concentration at 100 mg/L was determined. The initial concentration was 6.74 mg/L in the solution. However, the test item was not stable under the experimental conditions and concentration fell below 80 % of the initial value during the test (close to 0.1 mg/L after 48 hours, corresponding approximately to 1 % of the initial concentration). The concentration of the test item of 100 mg/L (nominal) corresponded to a geometric mean of measured concentrations over the 48-h test period of 0.518 mg/L.

It was assumed that the limit of solubility of the test item in M4 water (under our experimental conditions) was reached in the LS solution since the ratio between the loading rate used for its preparation (100 mg/L) and the initial measured concentration in this solution (6.74 mg/L) was >10. As no immobilization was observed at LS, the study was considered complete.

Hence, the highest concentration resulting in no immobilization at 48 hours was ≥ 100 mg/L expressed as initial loading rate, corresponding to 0.518 mg/L expressed as actual concentration (i.e. geometric mean of measured concentrations in the LS solution throughout the test).
The 48 -hour EC50 of the test item was > 100 mg/L expressed as initial loading rate, corresponding to 0.518 mg/L expressed as actual concentration (i.e. geometric mean of measured concentrations in the LS solution throughout the test).
Results with reference substance (positive control):
No reference substance
Reported statistics and error estimates:
Not performed

Immobilization - Limit test and range-finding test

Validity criteria fulfilled:
yes
Conclusions:
Under the experimental conditions, the 48-hour EC50 of tert-Butylperoxy-3,5,5-trimethylhexanoat in a static test system is > 100 mg/L expressed as initial loading rate, corresponding to 0.518 mg/L expressed as actual concentration (geometric mean of measured concentrations during the test in the saturated solution obtained from this loading rate of 100 mg/L) for Daphnia magna.
Executive summary:

Tert-Butylperoxy-3,5,5-trimethylhexanoat was tested for acute toxicity to Daphnia magna (clone 5) in a 48 -hour static test according to EU-method C.2 (1992) and OECD Test Guideline . The criterion measured is the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of the animals.

The stock solution, for the limit and range-finding tests, was prepared by dispersing the test item directly in M4 reconstituted water. The condition of preparation of the stock solution are reported as follows:

Quantity of test item: 100 mg

Volume of M4 reconstituted water: 1000 mL

Concentration of the test item: 100 mg/L

Duration of the agitation: 23h 50min

A loading rate of 100 mg/L was used to prepare the stock solution. As test item droplets were still in suspension after agitation, the stock solution was filtered through a filter of porosity 0.45 µm to remove the suspended fraction. The filtered solution, corresponding to the limit of solubility of the test item under the experimental conditions (LS), was then used to prepare the test solutions. Test solutions were prepared by further dilution of the LS solution with M4 reconstituted water to provide a geometric series of concentrations: 0 mg/L, LS/1000, LS/100, LS/10 and LS.

At the limit test twenty animals (in four replicates of five) were subjected to the LS solution (limit of solubility obtained from an initial loading rate of approximately 100 mg/L) and a further group of animals (in four replicates of five) was exposed to dilution water only (control), over a 48 -hour period. At the range-finding test three groups of 20 daphnids (in four replicates of five) were subjected to dilutions of the limit test solution at LS/1000, LS/100 and LS/10 for a period of 48 hours.

Under this experimental conditions, the 48 -hour EC50 of tert-Butylperoxy-3,5,5-trimethylhexanoat in a static system is >100 mg/L expressed as initial rate, corresponding to > 0.518 mg/L expressed as actual concentration (geometric mean of measured concentrations during the test in the saturated solution obtained from this loading rate of 100 mg/L) for Daphnia magna.

Description of key information

tert-Butylperoxy-3,5,5-trimethylhexanoat was assessed in a short-term toxicity to aquatic invertebrates study according to EU-method C.2 and OECD guideline 202 (1984). Daphnia were exposed in a static test to nominal concentrations of 0 mg/L, LS/1000, LS/100, LS/10 and LS (LS: limit of solubility obtained from an initial loading rate of 100 mg/L).
The 48 -hour EC50 of tert-Butylperoxy-3,5,5-trimethylhexanoat in a static system is >100 mg/L expressed as initial rate, corresponding to > 0.518 mg/L expressed as actual concentration (geometric mean of measured concentrations during the test in the saturated solution obtained from this loading rate of 100 mg/L) for Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
> 100 mg/L

Additional information

tert-Butylperoxy-3,5,5-trimethylhexanoat was assessed in a short-term toxicity to aquatic invertebrates study according to EU-method C.2 and OECD guideline 202 (1984). Daphnia were exposed in a static test to nominal concentrations of 0 mg/L, LS/1000, LS/100, LS/10 and LS (LS: limit of solubility obtained from an initial loading rate of 100 mg/L). The 48 -hour EC50 in a static system is >100 mg/L expressed as initial rate, corresponding to > 0.518 mg/L expressed as actual concentration (geometric mean of measured concentrations during the test in the saturated solution obtained from this loading rate of 100 mg/L) for Daphnia magna.