A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 01, 2004 to May 12, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Version / remarks:

Acute toxicity test, Third edition, 1996-04-01.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Protocol deviations:
1. During the range-finding test, vessel A in the solvent-control was found to contain only 4 daphnia instead of 5.
Evaluation: No effects in any but the highest test concentration.
2. During the final test, the difference between the lowest and highest measured concentration was slightly higher than 2°C, but this could be attributed to the relatively high temperature of the test medium prior to the start of the test.
The study integrity was not adversely affected by this deviation.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 18, 32, 56 and 100 mg/l and control

- Sampling method: at t= 0 h from freshly prepared solutions; at t= 21½ h from the freshly prepared solutions and the 21½ h-old solutions; at t=48 h from the 26½ h-old solutions. Volume 10 ml from the approximate centre of the test vessel.

- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A pretest was performed in order to determine the solubility in test medium. A weighed amount of 102.6 mg was added to 1 liter of ISO medium. After magnetic stirring overnight, the resulting solution was slightly hazy. After 4 hours of stabilisation, the solution was clear but contained precipitated test substance. A weighed amount of 10.034 mg was added to 1 liter of ISO-medium. Magnetic stirring or treatment with ultrasonic waves did not accelerate the dissolution of the test substance. Finally, acetone and methanol were tested as presolvents. Weighed amounts of 100.4 and 99.8 mg were added to 1 ml acetone and methanol, respectively. After vigorous stirring, the test substance was completely dissolved in both solvents. Subsequently, 100 µl of the solution in acetone was added to 1 liter ISO-medium, resulting in a clear and colourless solution. Finally, a weighed amount of 0.9993 mg was added to 1 ml acetone and vigorously stirred for 30 minutes, after which it was completely dissolved. 200 µl of this solution was added to 1 liter ISO medium, resulting in a clear and colourless solution with test substance droplets. Magnetic stirring overnight resulted in a clear and colourless solution with precipitating test substance droplets.

In the static range-finding test, preparation of test solutions started with a stock solution of 100 mg/ml in acetone (Pestiscan 99.8%, Labscan, Ireland). This stock was diluted in acetone to reach concentrations a factor 10,000 above the target concentrations. Amounts of 50 µl were added to 500 ml ISO-medium under magnetic stirring, and the resulting solutions were all clear and colourless.

In the semi-static final test, preparation of test solutions started with separately prepared stock solutions in acetone (Pestiscan 99.8%, Labscan, Ireland) at concentrations a factor of 10,000 above the target concentrations in test medium. These solutions were then added to ISO-medium. After 48 hours of magnetic stirring, the solutions were all clear and colourless, except for the highest two concentrations, which were clear but contained some floating particles. After 24 hours of stabilisation and siphoning, the test solutions were all clear and colourless. Note that test solutions were renewed after 21½ hours. Since all solutions were siphoned, they are referred to as Water Accommodated Fractions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna
- Source: In-house laboratory culture with a known history
- Age at study initiation (mean and range, SD): For the test selection of young daphnia with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Method of breeding: acyclical parthenogenesis under specified breeding conditions
- Feeding during test: no feeding

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observations

Hardness:

250 mg/l expressed as CaCO3

Test temperature:

18.7 - 20.3 °C

pH:

7.5 - 7.8

Dissolved oxygen:

8.6 - 9.1 mg/l

Nominal and measured concentrations:

Loading rate 10 mg/l; t=0 h: Measured concentration: 10.8 mg/l
Loading rate 10 mg/l; t=21.5 h old: Measured concentration: 10.4 mg/l
Loading rate 10 mg/l; t=21.5 h fresh: Measured concentration: 9.23 mg/l
Loading rate 10 mg/l; t=48 h: Measured concentration: 9.05 mg/l

Loading rate 18 mg/l; t=0 h: Measured concentration: 15.6 mg/l
Loading rate 18 mg/l; t=21.5 h old: Measured concentration: 15.2 mg/l
Loading rate 18 mg/l; t=21.5 h fresh: Measured concentration: 15.8 mg/l
Loading rate 18 mg/l; t=48 h: Measured concentration: 15.6 mg/l

Loading rate 32 mg/l; t=0 h: Measured concentration: 30.1 mg/l
Loading rate 32 mg/l; t=21.5 h old: Measured concentration: 27.5 mg/l
Loading rate 32 mg/l; t=21.5 h fresh: Measured concentration: 23.4 mg/l
Loading rate 32 mg/l; t=48 h: Measured concentration: 22.9 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass
- Aeration: No aeration of the test solutions.
- Renewal rate of test solution (frequency/flow rate): Renewal of test solutions after 21½ hours.
- No. of organisms per vessel: 5 per vessel containing 80 ml medium
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium
- Culture medium different from test medium: yes (M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33)).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

9.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Other biological observations: The responses recorded in this test allowed for reliable determination of an EC50. The responses recorded at 10 mg/l were in agreement with the results of the range-finding test. At the end of the test, 90% of the daphnids exposed to a loading rate of 32 mg/l were immobilised, and 100% of the daphnia exposed to WAFs prepared at loading rates of 56 and 100 mg/l were immobilised.

- Mortality of control: no daphnia became immobilised in control treatment

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Immobility after 24h: 0% at blank control; 0% at 0.10; 0% at 0% at 0.18, 0% at 0.32, 0% at 0.56, 0% at 1.0 and 100% at 1.8 mg/l.
Immobility after 48h: 0% at blank control; 0% at 0.10; 0% at 0% at 0.18, 0% at 0.32, 0% at 0.56, 90% at 1.0 and 100% at 1.8 mg/l.

- EC50/LC50:
The 24h-EC50 was 1.3 mg/l with 0% immobilisation at 1.0 mg/l and complete immobilisation at 1.8 mg/l.
The 48h-EC50 was 0.80 mg/l with a 95% confidence interval between 0.73 and 0.94 mg/l.

Reported statistics and error estimates:

Average exposure concentrations were calculated as the geometric means of the measured concentrations at the start (Ct=0 and Ct=21.5) and the end (Ct=21.5 and Ct=48) of the two renewal periods.
Calculation of EC50:
The EC50-value was calculated at 21½ and 48 hours of exposure from the probits of the percentages of affected daphnia and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, the test substance did not induce acute immobilisation of Daphnia magna at 9.9 mg/l after 48 hours of exposure (NOEC). Based on loading rates, the NOEL was 10 mg/l. The 48h EC50 was 19 mg/l based on average exposure concentrations (95% confidence interval between 17 and 22 mg/l). Based on loading rates, the EC50 was 22 mg/l with a 95% confidence interval between 19 and 26 mg/l.

Executive summary:

A study was conducted to determine the acute toxicity of the test substance in aquatic invertebrates (Daphnia magna) according to OECD Guideline 202, EU Method C.2 and ISO 6341, in compliance with GLP. Following an initial range-finding study, the main experiment was performed under semi-static conditions. Preparation of test solutions started with separately prepared stock solutions in acetone at concentrations a factor of 10,000 above the target concentrations in test medium. These solutions were then added to ISO-medium. After 48 h of magnetic stirring followed by 24 h of stabilisation and siphoning, the test solutions were all clear and colourless. The test solutions were renewed after 21 h. Twenty daphnia per concentration (in quadruplicate, 5 per vessel) were exposed to loading rates of 0, 10, 18, 32, 56 and 100 mg/L. The total test period was 48 h. Samples for analysis were taken at the start and at the end of the two renewal periods. Analysis of the samples taken showed that measured concentrations were in agreement with nominal concentrations. Test concentrations remained fairly stable during the renewal periods. The results were based on average exposure concentrations as well as on the loading rates. The test met the acceptability criteria prescribed by the protocol and was considered valid. At the end of the test, 90% of the daphnids exposed to a loading rate of 32 mg/L were immobilised, and 100% of the daphnia exposed to water accommodated fractions prepared at loading rates of 56 and 100 mg/L were immobilised. The test substance did not induce acute immobilisation of Daphnia magna up to 9.9 mg/L after 48 h of exposure. Under the study conditions, the 48 h EC50 of the test substance in Daphnia magna was determined to be 19 mg/L based on average exposure concentrations (de Roode, 2004).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

19 mg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance in aquatic invertebrates (Daphnia magna) according to OECD Guideline 202, EU Method C.2 and ISO 6341, in compliance with GLP. Following an initial range-finding study, the main experiment was performed under semi-static conditions. Preparation of test solutions started with separately prepared stock solutions in acetone at concentrations a factor of 10,000 above the target concentrations in test medium. These solutions were then added to ISO-medium. After 48 h of magnetic stirring followed by 24 h of stabilisation and siphoning, the test solutions were all clear and colourless. The test solutions were renewed after 21 h. Twenty daphnia per concentration (in quadruplicate, 5 per vessel) were exposed to loading rates of 0, 10, 18, 32, 56 and 100 mg/L. The total test period was 48 h. Samples for analysis were taken at the start and at the end of the two renewal periods. Analysis of the samples taken showed that measured concentrations were in agreement with nominal concentrations. Test concentrations remained fairly stable during the renewal periods. The results were based on average exposure concentrations as well as on the loading rates. The test met the acceptability criteria prescribed by the protocol and was considered valid. At the end of the test, 90% of the daphnids exposed to a loading rate of 32 mg/L were immobilised, and 100% of the daphnia exposed to water accommodated fractions prepared at loading rates of 56 and 100 mg/L were immobilised. The test substance did not induce acute immobilisation of Daphnia magna up to 9.9 mg/L after 48 h of exposure. Under the study conditions, the 48 h EC50 of the test substance in Daphnia magna was determined to be 19 mg/L based on average exposure concentrations (de Roode, 2004).