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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 29, 2004 to June 10, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Protocol deviations:
1. At the end of the range-finding test, pH was measured in all solutions. The pH in the controls and the two lowest concentrations was above 9 at the end of the test, and the pH in the blank-control had increased by more than 1.5 unit from the value measured at the start of the test.
Evaluation: This was related to relatively high rates of algal growth.
2. The pH in the blank-control in the second EC50 test increased by more than 1.5 unit and was above 9 at the end of the test.
Evaluation: This was related to relatively high rates of algal growth.

The study integrity was not adversely affected by the deviations.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

First EC50 test
- Concentrations: 3.0, 6.6, 15, 32 and 70 mg/l, blank control and solvent control
- Sampling method: 10 ml at t=0 h, t=24 h and t=72 h
- Sample storage conditions before analysis: freezer

Second EC50 test
- Concentrations: Nominal concentrations of 3.0 and 6.6 mg/l and WAFs prepared at loading rates of 15, 32 and 70 mg/l, blank control and solvent control
- Sampling method: 10 ml at t=0 h, t=24 h and t=72 h
- 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. 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 range-finding test, preparation of test solutions of 10 mg/l and below started with stock solutions in acetone (Pestiscan 99.8%, Labscan, Ireland) at a factor 10,000 above the target concentrations. Amounts of 50 µl were added to 500 ml M2 medium, and the resulting solutions were all clear and colourless after 30 minutes of magnetic stirring. The highest test concentration was prepared using a stock of 100 mg test substance in 100 mg acetone, which was added to 1 liter M2 medium. Following 25 hours of magnetic stirring, this solution was clear and colourless.

In the first EC50 test, preparation of test solutions started with stock solutions separately prepared in acetone (Pestiscan 99.8%, Labscan, Ireland). Weighed amounts of test substance were mixed with 100 mg acetone. These solutions were then added to M2 medium and magnetically stirred for 48 hours. Subsequently, the solutions were stabilised for 24 hours. The WAFs were taken and used for testing. These WAFs were all clear and colourless.

In the second EC50 test, preparation of test solutions started with stock solutions in acetone (Pestiscan 99.8%, Labscan, Ireland): the stocks for the two lowest test concentrations were prepared at a factor of 10,000 above the target concentration, of which 50 µl was added to 500 ml M2 medium. The three highest concentrations were all prepared in 100 mg acetone, which was then added completely to 1 liter M2 medium. Following 20½ hours of magnetic stirring, the two lowest concentrations were clear and colourless, while the highest three concentrations contained precipitate. The WAFs taken from these solutions were all clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 104 cells/ml. In the EC50 tests, glassware was silanised prior to use: after rinsing three times with 2% dichlorodimethylsilane in heptane and drying under pressure air, the glassware was rinsed three times with milli-RO water and dried in an incubator at 85°C.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): not indicated
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.


ACCLIMATION
- Acclimation period: 3 days before the start of the test
- Culturing media and conditions (same as test or not):
Stock culture media was M1; formulated using Milli-Ro water (tap-water purified by reverse osmosis).
Test medium and pre-culture medium was M2; according to the ISO-Standard "Algal growth inhibition test" Nov. 1989; formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges) preventing precipitation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observations.

Hardness:

24 mg CaCO3/l)

Test temperature:

First experiment: 23.5 - 23.8°C
Second experiment: 23.6 and 23.9°C

pH:

First experiment: 7.1 - 8.2
Second experiment: 7.0 - 9.4

Nominal and measured concentrations:

First experiment:
Loading rate 3.0 mg/l t=TWA: Measured concentration = 2.6 mg/l (TWA=time weighed average )
Loading rate 6.6 mg/l t=TWA: Measured concentration = 3.2 mg/l
Loading rate 15 mg/l t=TWA: Measured concentration = 7.9 mg/l
Loading rate 32 mg/l t=TWA: Measured concentration = 21 mg/l
Loading rate 70 mg/l t=TWA: Measured concentration = 41 mg/l

Second experiment:
Loading rate 3.0* mg/l t=average: Measured concentration = 2.6 mg/l
Loading rate 6.6* mg/l t=average: Measured concentration = 5.1 mg/l
Loading rate 15 mg/l t=average: Measured concentration = 11 mg/l
Loading rate 32 mg/l t=average: Measured concentration = 27 mg/l
Loading rate 70 mg/l t=average: Measured concentration = 44 mg/l
* nominal concentrations (no WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, silanised, containing 50 ml of test medium (1st and 2nd experiment)

- Initial cells density: An initial cell density of 10000 cells/ml. (1st and 2nd experiment)
- Control end cells density: First expetiment: 839000 cells/ml
Second experiment: 915000 cells/ml
-Replicates:
First experiment:
3 replicates of each test concentration.
6 replicates of the controls.
1 replicate of each test concentration without algae.
2 additional replicates of the highest concentration without algae
1 additional replicate of each test concentration and control for sampling purposes

Second experiment:
3 replicates of each test concentration.
6 replicates of the controls.
2 replicates of the highest concentration without algae
1 additional replicate of each test concentration and control for sampling purposes.

GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 74 to 104 uE.m-2.s-1.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
First test:
At the beginning of the test, cells were counted using a microscope and a counting chamber.
The test solutions were so turbid that they disturbed spectrophotometric measurement. Therefore algal density was determined by use of a microscope and a counting chamber throughout the test.

Second test:
At the beginning of the test, cells were counted using a microscope and a counting chamber.
Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Varian Nederland BV., Houten, The Netherlands. Algal medium was used as blank. After 72 hours, the test solutions were so turbid that they disturbed spectrophotometric measurement. Therefore algal density was also determined by use of a microscope and a counting chamber at the end of the test.


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
First test: WAFs prepared at loading rates of 3.0, 6.6, 15, 32 and 70 mg/l.
Second test: Nominal concentrations of 3.0 and 6.6 mg/l and WAFs prepared at loading rates of 15, 32 and 70 mg/l.

- Range finding study: the study is a combined limit/range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg/l and to a blank- and a solvent-control
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and cell number

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities
- Any stimulation of growth found in any treatment: no

- Other: The responses in the second EC50 test were more comparable to those of the range-finding test than the responses in the first EC50 test. Therefore, the conclusion will be based on the results of this second EC50 test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: The EC50 for cell growth inhibition (EBC50: 0-72h) was 0.49 mg/l with a 95% confidence interval ranging from 0.28 to 0.85 mg/l.
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.0 mg/l with a 95% confidence interval ranging from 0.58 to 1.8 mg/l.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline (201, adopted 7 June 1984) were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth or inhibition of growth rate (ANOVA, Tukey test, Bonferroni t-test).
Calculation of the EC/EL50 and EC/EL10 values was based on log-linear regression analysis of the percentages of growth inhibition and the percentages of growth rate reduction versus the logarithms of the corresponding average exposure concentrations or loading rates of the test substance.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Selenastrum capricornutum, test substance reduced growth rate of this fresh water algae species significantly at TWA concentrations of 11 mg/l and higher. The EC50 for cell growth inhibition (EBC50: 0-72h) was 13 mg/l based on TWA concentrations. Based on loading rates, the EL50 for cell growth inhibition (EBL50) was 18 mg/l. The EC50 for growth rate reduction (ERC50: 0-72h) was 33 mg/l, based on TWA concentrations. Based on loading rates, the EL50 for growth rate reduction (ERL50) was 47 mg/l. The NOEC for both cell growth inhibition and growth rate reduction was a TWA concentration of 5.1 mg/l. Based on loading rates, the NOEL was 6.6 mg/l.

Executive summary:

A study was conducted to determine the short term toxicity of the test substance to aquatic algae according to OECD Guideline 201, EU Method C.3 and ISO 8692, in compliance with GLP. A pretest was performed in order to determine the solubility in test medium. Two valid experiments were performed with the algae Selenastrum capricornutum. The first study was conducted using 5 concentrations ranging from 3.0 to 70 mg/L, with incubation time of 72 h. In the second study nominal concentrations of 3.0 and 6.6 mg/L were tested and water accommodated fractions (WAFs) were prepared at loading rates of 15, 32 and 70 mg/L. The cell concentration of each replicate was determined by measuring the cell number every 24 h. The growth rate and the yield were determined from the cell number at the respective observation time. In the present study, test substance reduced growth rate of this fresh water algae species significantly at time weighted average (TWA) concentrations of 11 mg/L and higher. The 72 EC50 for cell growth inhibition (EbC50) was determined to be 13 mg/L based on TWA concentrations. Based on loading rates, the EL50 for cell growth inhibition (EbL50) was 18 mg/L. The 72 h ErC50 for growth rate reduction was determined to be 33 mg/L, based on TWA concentrations. Based on loading rates, the EL50 for growth rate reduction (ErL50) was 47 mg/L. The NOEC for both cell growth inhibition and growth rate reduction was determined to be a TWA concentration of 5.1 mg/L (de Roode, 2004).

Key value for chemical safety assessment

EC50 for freshwater algae:

33 mg/L

EC10 or NOEC for freshwater algae:

5.1 mg/L

Additional information

A study was conducted to determine the short term toxicity of the test substance to aquatic algae according to OECD Guideline 201, EU Method C.3 and ISO 8692, in compliance with GLP. A pretest was performed in order to determine the solubility in test medium. Two valid experiments were performed with the algae Selenastrum capricornutum. The first study was conducted using 5 concentrations ranging from 3.0 to 70 mg/L, with incubation time of 72 h. In the second study nominal concentrations of 3.0 and 6.6 mg/L were tested and water accommodated fractions (WAFs) were prepared at loading rates of 15, 32 and 70 mg/L. The cell concentration of each replicate was determined by measuring the cell number every 24 h. The growth rate and the yield were determined from the cell number at the respective observation time. In the present study, test substance reduced growth rate of this fresh water algae species significantly at time weighted average (TWA) concentrations of 11 mg/L and higher. The 72 EC50 for cell growth inhibition (EbC50) was determined to be 13 mg/L based on TWA concentrations. Based on loading rates, the EL50 for cell growth inhibition (EbL50) was 18 mg/L. The 72 h ErC50 for growth rate reduction was determined to be 33 mg/L, based on TWA concentrations. Based on loading rates, the EL50 for growth rate reduction (ErL50) was 47 mg/L. The NOEC for both cell growth inhibition and growth rate reduction was determined to be a TWA concentration of 5.1 mg/L (de Roode, 2004).