1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

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:

2017-12-29 to 2018-01-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Analytical purity: 98.2%
- Source and lot/batch No.of test material: I16FD3167
- Expiration date of the lot/batch: 2018-10-26 (retest date)
- Purity test date: 2018-08-27 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: solubility in water: Not stable

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples were taken before the start of the test and after 72 hours from each test medium and from the control. Additionally, reserve samples of 2.0 mL were taken for possible analysis. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In the combined limit/range-finding test, preparation of test solutions started with the highest test concentration of 100 mg/L applying 113 minutes of magnetic stirring at room temperature to accelerate the dissolution of the test item in test medium. The lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium.
In the final test, preparation of test solutions started with a loading rate of 100 mg/L applying 181 minutes of magnetic stirring at room temperature followed by 10 minutes of ultrasonic waves to ensure maximum dissolution of the test item in medium. This resulted in a clear and colourless dispersion that contained undissolved floating material. The obtained mixture was filtered through a 0.45 µm membrane filter (Whatman; RC55) to remove the undissolved fraction. The filter was pre-conditioned with a small volume of test solution that was discarded. The resulting saturated solution (SS) was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal 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 21-24°C. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

ACCLIMATION
- Pre-culture 3 days before start of the test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21-23 °C

pH:

t=0h: 6.5-8.2
t=72h: 7.8-8.5

Dissolved oxygen:

not applicable

Salinity:

not relevant

Conductivity:

not reported

Nominal and measured concentrations:

Range-finder:
nominal test concentrations: Control, 0.1, 1.0, 10 and 100% SS (0.1 and 1.0% SS not analysed)
measured test concentration t = 0 h: 8.80, 90.2 mg/L
measured test concentration t = 24 h: 8.73, 89.5 mg/L
measured test concentration t = 72 h: 8.68, 87.3 mg/L

Final test:
nominal test concentrations: Control, 1.0, 3.2, 10, 32 and 100% of a SS prepared at 100 mg/L
measured test concentration t = 0 h: n.d., 0.933, 2.99, 9.36, 9.78, 28.6, 94.4 mg/L
measured test concentration t = 72 h: n.d., 0.928, 3.05, 9.72, 9.68, 28.9, 96.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Initial cells density: 10,000 cells/mL
- Control end cells density: 360.3 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: yes, the obtained filtrate of the 100% saturated solution was set with 1M HCl from 9.1 to 8.0 in the combined range finder/limit test and from 9.3 to 8.3 in the final test
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps (with a light intensity within the range of 86 µE.m-2.s-1)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: At the beginning, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength = 10mm).
- Effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations: 0.1, 1.0, 10 and 100% of the SS (setup as combined range finder/limit test)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on biological relevance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on statistical significance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: based on statistical significance and biological relevance

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

- 72-h EC50 (growth rate) > 100 mg T002632/L
- 72-h EC50 (yield) >100 mg T002632/L

- 72-h EC10 (growth rate) >100 mg T002632/L
- 72-h EC10 (yield) = not determined

- 72-h NOEC (growth rate) = 32 mg T002632/L (based on statistical significance)
- 72-h NOEC (growth rate) = 100 mg T002632/L (based on biological relevance)
- 72-h NOEC (yield) = 32 mg T002632/L (based on statistical significance and biological relevance)

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-h EC50 (growth rate) = 0.86 mg/L (95% C.L. 0.84 to 0.88 mg/L)
- 72-h EC50 (yield) = 0.34 mg/L (95% C.L. 0.34 to 0.35 mg/L)

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 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 control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential T-Test, α=0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum concentration tested).
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T002632 according to the OECD guideline 201 (GLP conditions). The determined 72h-EC50 for both growth rate and yield inhibitoin were beyond the range tested, i.e. exeeded an analytically confirmed nominal concentration of 100 mg/L. The 72h-NOEC for growth rate was 32 mg/l based on statistical significance and 100 mg/L based on biological relevance. The 72h-NOEC for yield inhibition was 32 mg/L based on both statistical significance and biological relevance. The results of the test can be considered reliable without restrictions.

Description of key information

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T002632 according to the OECD guideline 201. The determined 72h-EC50 for both growth rate and yield inhibitoin were beyond the range tested, i.e. exeeded an analytically confirmed nominal concentration of 100 mg/L. The 72h-NOEC for growth rate was 32 mg/l based on statistical significance and 100 mg/L based on biological relevance. The 72h-NOEC for yield inhibition was 32 mg/L based on both statistical significance and biological relevance.