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 fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 01, 2004 to May 14, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Principles of method if other than guideline:

Protocol deviations:
After all fish had died at 10 mg/l, samples were taken upon removal of the fish, instead of before removal.
Evaluation: since all concentrations were in agreement with nominal, the concentration just before removal of the fish is expected to be sufficiently represented by these samples. The study integrity was not adversely affected by this deviation.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 1.8, 3.2, 5.6 and 10 mg/l
- Sampling method: 10 ml at t=0 h and t=72 h from freshly prepared solutions and at t=24 h and t=96 h from the 24h-old solutions.
- Sample storage conditions before analysis: freezer

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. An amount of 102.6 mg of the viscous liquid was weighed in a glass 20 ml vial, which was broken and 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.
An amount of 10.034 mg was weighed on the lid of an eppendorph cup and then 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 25-30 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 of 10 mg/l and below 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 500 µl were added to 5000 ml ISO-medium, and the resulting solutions were all clear and colourless. The highest test concentration was prepared using a stock of 500 mg test substance in 500 mg acetone, which was added to 5 liters ISO-medium. Following 24 hours of magnetic stirring and 2 hours of stabilisation, this solution was clear but contained precipitate. The Water Accommodated Fraction (WAF) was siphoned off and used as such.

In the semi-static final test, preparation of test solutions started with a serial dilution of a stock prepared in acetone (Pestiscan 99.8%, Labscan, Ireland) at concentrations a factor of 10,000 above the target concentrations in test medium (500 µl of the respective stock solution was added to 5 litres of ISO-medium). After 48 hours of magnetic stirring, the solutions were all clear and colourless. Following a 24 hour stabilisation period, the test solutions were all clear and colourless. Note that test solutions were renewed daily.

- Controls:
Test medium without test substance but with acetone used in the treatment of the stock solutions (solvent-control).

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Strain: Cyprinus carpio
- Source: Zodiac, proefacc, "De Haar Vissen", L.U. Wageningen, the Netherlands.
- Age at study initiation (mean and range, SD): not indicated
- Length at study initiation (length definition, mean, range and SD): 2.3 ± 0.1 cm
- Weight at study initiation (mean and range, SD): 0.36 ± 0.09 g
- Method of breeding: F1 from a single parent-pair bred in UV-treated water
- Feeding during test : No feeding from 48 hours prior to the test and during the total test period.


ACCLIMATION
- Acclimation period: At least 12 days after delivery

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observations

Hardness:

250 mg CaCO3 per litre

Test temperature:

21.0 - 21.7

pH:

7.2 - 7.9

Dissolved oxygen:

6.0 - 9.0 mg/l

Nominal and measured concentrations:

Loading rate 0 mg/l t=0h : n.d.
Loading rate 3.2 mg/l t=0h : 2.98 mg/l
Loading rate 5.6 mg/l t=0h : 5.10 mg/l
Loading rate 10 mg/l t=0h : 9.02 mg/l

Loading rate 0 mg/l t=24h : n.d.
Loading rate 3.2 mg/l t=24h : 2.88 mg/l
Loading rate 5.6 mg/l t=24h : 4.85 mg/l
Loading rate 10 mg/l t=24h : 8.62 mg/l

Loading rate 0 mg/l t=72h : n.d.
Loading rate 3.2 mg/l t=72h : 2.95 mg/l
Loading rate 5.6 mg/l t=72h : 4.91 mg/l

Loading rate 0 mg/l t=96h : n.d.
Loading rate 3.2 mg/l t=96h : 2.75 mg/l
Loading rate 5.6 mg/l t=96h : 4.52 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 6 litres, all-glass, containing 5 litres of test medium
- Aeration: The test media were not aerated during the test.
- Renewal rate of test solution (frequency/flow rate): renewal of test solutions after each 24-hour test period.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.50 g fish/litre, i.e. 7 fish per 5 litres of test medium


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-medium, formulated using Milli-Ro water (tap-water purified by reverse osmosis) with the following composition:
CaCl2.2H2O: 293.8 mg/l
MgSO4.7H2O: 123.3 mg/l
NaHCO3: 64.8 mg/l
KCl: 5.8 mg/l

OTHER TEST CONDITIONS
- Adjustment of pH: 7.8-8.4.
- Photoperiod: 16 hours photoperiod daily
- Light intensity: not indicated

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.1, 1.0 and 10 mg/l and WAF (Water Accomodating Fraction) prepared at a loading rate of 100 mg/l

Reference substance (positive control):

yes

Remarks:

pentachlorophenol

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities:
Solvent-control, t=0-96h: no abnormalities (relative number 7/7)
1.0 mg/l, t=0-96h: no abnormalities (relative number 7/7)
1.8 mg/l, t=0-96h: no abnormalities (relative number 7/7)
3.2 mg/l, t=0-96h: no abnormalities (relative number 7/7)
5.6 mg/l, t=0-24h: no abnormalities (relative number 7/7)
5.6 mg/l, t=48h: Swimming at the surface (relative number 5/6)
5.6 mg/l, t=48h: Swimming at the bottom (relative number 1/6)
5.6 mg/l, t=72h: Swimming at the surface (relative number 2/3)
5.6 mg/l, t=72h: Immobile (relative number 1/3)
5.6 mg/l, t=96h: Visible damage on fins (relative number 1/2)
10 mg/l, t=0-2h: No abnormalities (relative number 7/7)
10 mg/l, t=024h: Discoloured (relative number 6/7)
10 mg/l, t=024h: Discoloured and snapping at the surface (1/7)

- Mortality of control: no mortality and no sublethal effects observed

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality: 0% at 0.10 mg/l; 0% at 0.22 and 0.46 mg/l
- LC50: 0.32 mg/l (95 % confidence interval between 0.22 and 0.46 mg/l)

Reported statistics and error estimates:

The LC50 was determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Validity criteria fulfilled:

yes

Remarks:

These conditions all remained within the limits prescribed by the protocol

Conclusions:

Under the conditions of the present study, the test substance induced no visible effects in carp at or below 3.2 mg/l (NOEC). The 96h LC50 was 4.9 mg/l based on analytically confirmed nominal concentrations (95% confidence interval between 4.4 and 6.4 mg/l).

Executive summary:

A study was conducted to determine the acute toxicity of the test substance in fish according to OECD Guideline 203, EU Method C.1 and ISO 7346-1, in compliance with GLP. Following a preliminary range-finding study, a semi-static main experiment was conducted. 7 fish (Cyprinus carpio) per concentration was exposed to the test substance at concentrations of 0, 1.0, 1.8, 3.2, 5.6 and 10 mg/L. Preparation of test solutions started with a serial dilution of a stock prepared in acetone. After 48 h of magnetic stirring, the solutions were all clear and colourless. Following a 24 h stabilisation period, the test solutions were all clear and colourless. Test solutions were renewed daily. The total test period was 96 h. Samples for analysis were taken at the start and at the end of the first and the last renewal period. Analysis of the samples taken during the final test showed that measured concentrations were in agreement with nominal concentrations throughout the test period by 81 -93%. Therefore, results were based on nominal concentrations. The test met the acceptability criteria prescribed by the protocol and was considered valid. The test substance induced no visible effects in the fish up to 3.2 mg/L. Under the study conditions, the 96 h LC50 of the test substance in fish was determined to be 4.9 mg/L based on analytically measured concentrations (de Roode, 2004).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.9 mg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance in fish according to OECD Guideline 203, EU Method C.1 and ISO 7346-1, in compliance with GLP. Following a preliminary range-finding study, a semi-static main experiment was conducted. 7 fish (Cyprinus carpio) per concentration was exposed to the test substance at concentrations of 0, 1.0, 1.8, 3.2, 5.6 and 10 mg/L. Preparation of test solutions started with a serial dilution of a stock prepared in acetone. After 48 h of magnetic stirring, the solutions were all clear and colourless. Following a 24 h stabilisation period, the test solutions were all clear and colourless. Test solutions were renewed daily. The total test period was 96 h. Samples for analysis were taken at the start and at the end of the first and the last renewal period. Analysis of the samples taken during the final test showed that measured concentrations were in agreement with nominal concentrations throughout the test period by 81 -93%. Therefore, results were based on nominal concentrations. The test met the acceptability criteria prescribed by the protocol and was considered valid. The test substance induced no visible effects in the fish up to 3.2 mg/L. Under the study conditions, the 96 h LC50 of the test substance in fish was determined to be 4.9 mg/L based on analytically measured concentrations (de Roode, 2004).