Triethyl O-acetylcitrate

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:

2010-05-20 - 2010-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material was used.

Analytical monitoring:

yes

Details on sampling:

- Sampling method/ concentrations: Approximately 20 mg test item were diluted with 20 mL of a mixture of acetonitrile / pure water (50/50) to obtain stock solutions of 1 g test item/L. Appropriate amounts of these stock solutions were diluted with test water to obtain fortified samples at concentrations of 10 and 100 mg test item/L.
- Sample storage conditions before analysis: All samples were stored in a freezer (< - 10 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: Test water without addition of the test item
Stock Solution: The test item was used to prepare a stock solution. Approximately 20 mg of test item were dissolved in 20 mL of a mixture of acetonitrile / pure water (50/50) to obtain a stock solution of 1 g test item/L.
Standard Solutions: Appropriate amounts of the stock solution were diluted with a mixture of pure water/acetonitrile (50/50) to obtain standard solutions in the range from 2.5 to 75 mg test item/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Strain No. 61.81 SAG
- Source: The algae were supplied by "The Collection of Algal Cultures" of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Breeding Conditions: The algae were cultivated in the laboratories of the testing facility under standardised conditions according to the test guidelines.

PREPARATION OF PRE-CULTURES
Pre-cultures were set up three days prior to the test start under the same conditions as in the test.

TEST CULTURES
The test was started (0 hours) by inoculation of a biomass of 5000 algal cells per mL test medium. These cells were taken from the pre-culture.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period is described.

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.

Test temperature:

The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. Water temperature was 23 - 24 °C.

pH:

The pH was measured in all test item concentrations and the control at the start and the end of the test. pH was 8.2 at test start and 9.6 at test end.

Dissolved oxygen:

No details available.

Salinity:

No details available.

Nominal and measured concentrations:

The only concentration tested was nominal 100 mg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume
- Type: closed
- Initial cells density: approximately 5000 cells per millilitre
- No. of vessels per concentration (replicates): six
- No. of vessels per control (replicates): six
- Other: Blanks: Additionally, one replicate of the only test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometrical measurements. The additional replicates were incubated under same conditions.
- Other: Cell density measurements: The cell densities were determined by spectrophotometrical measurement. Therefore defined volumes of the
algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (OECD medium); analytical grade salts were added in deionised water (conductivity <5 µScm-1). Macro-nutrients are NaHCO3, CaCl2*2 H2O, NH4Cl, MgSO4*7 H2O, MgCl2*6 H2O and KH2PO4.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was adjusted with 1 M NaOH to pH 8.2.
- Photoperiod: Continuous illumination
- Light intensity and quality: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media. Mean light intensity = 4785 lux (range: 4580-5180 lux).

TEST CONCENTRATIONS
The only test concentration of 100 mg test item/L was prepared by dissolving 100 mg test item into 1000 mL test water by intense stirring for 90 minutes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Growth Inhibition: The 72-hour NOEC was determined to be at least nominal 100 mg test item/L (mean measured: at least 79 mg test item/L). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 72-hour LOEC and the 72-hour EC50 were clearly higher than nominal 100 mg test item/L (mean measured: clearly higher than 79 mg test item/L). These values could not be quantified due to the absence of toxicity of the test item up to nominal 100 mg test item/L..
Microscopic Examination: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing at a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the only concentration tested.
Appearance of the test item in test water: There were no remarkable observations.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate p.a. is tested at least twice a year to demonstrate satisfactory test conditions, but no results or further details are given.

Reported statistics and error estimates:

For the determination of the 72-hour LOEC and the 72-hour NOEC the calculated growth rates and yields at the only test concentration were tested for significant differences compared to the control values by the Student's t- test (growth rate) and the Welch t-test (yield), respectively. The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat® Solutions GmbH.

Validity criteria of the study

Cell density increase in control cultures: 205-fold increase within 72 hours and thus, validity criterion was met.

Coefficient of variation of sectional (daily) growth rates in control cultures: 33 % and thus, validity criterion was met.

Coefficient of variation of average growth between control replicates: 1.3 % and thus, validity criterion was met.

Analytical results

At the start of the test 101 % of the nominal test concentrations were found (average of the only test concentration). After 72 hours test duration, 62 % of the nominal value was determined (average of the only test concentration). During the test the algae were exposed to a geometrical mean of 79 % of nominal. Therefore, all reported results refer to the measured geometrical mean.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria of the test method were met.

Conclusions:

The 72-hour NOEC was determined to be at least nominal 100 mg test item/L (mean measured: at least 79 mg test item/L). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 72-hour LOEC and the 72-hour EC50 were clearly higher than nominal 100 mg test item/L (mean measured: clearly higher than 79 mg test item/L). These values could not be quantified due to the absence of toxicity of the test item up to nominal 100 mg test item/L.

Executive summary:

The acute toxicity to aquatic algae and cyanobacteria of 'triethyl O-acetylcitrate' was tested according to OECD guideline 201 and EU Method C.3 in an Algal Growth Inhibition Limit Test with the freshwater green algae Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) as test organism (Boettcher M., Wydra V., 2010). The study was conducted under certificated GLP compliance. The only concentration tested was nominal 100 mg test item/L. Additionally, a control was tested in parallel (test water without addition of the test item). Thus a limit test was performed in accordance with the OECD 201 to demonstrate that the test item has no inhibitory effect on the growth of the test organism up to this concentration. The study encompassed 2 treatment groups (1 dose rate of the test item, control) with six replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test. All calculations were carried out using a statistical software programme (ToxRatPro Version 2.10). Analysis of the yield and growth rate of the algal population within 72 h exposure period gave the following results:

The NOE_rC (72h) and the NOE_yC (72h) were determined to be at least nominal 100 mg test item/L. The NOEC values might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The LOE_rC (72h), the LOE_yC (72h), the E_rC50 (72h) and the E_yC50 were clearly higher than nominal 100 mg test item/L (mean measured: clearly higher than 79 mg test item/L). No toxic effects against algae were observed at the limit of water solubility under these exposure conditions.

Description of key information

Pseudokirchnerella subcapitata (reported as Selenastrum capricornutum):
Effect based on growth rate: ErC50 (72h) > 100 mg/L, NOErC ≥ 100 mg/L, LOErC > 100 mg/L
Effect based on yield: EyC50 (72h) > 100 mg/L, NOEyC ≥ 100 mg/L, LOEyC > 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The acute toxicity to aquatic algae and cyanobacteria of 'triethyl O-acetylcitrate' was tested according to OECD guideline 201 and EU Method C.3 in an Algal Growth Inhibition Limit Test with the freshwater green algae Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) as test organism (Boettcher M., Wydra V., 2010). The study was conducted under certificated GLP compliance. The only concentration tested was nominal 100 mg test item/L. Additionally, a control was tested in parallel (test water without addition of the test item). Thus a limit test was performed in accordance with the OECD 201 to demonstrate that the test item has no inhibitory effect on the growth of the test organism up to this concentration. The study encompassed 2 treatment groups (1 dose rate of the test item, control) with six replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test. All calculations were carried out using a statistical software programme (ToxRatPro Version 2.10). Analysis of the yield and growth rate of the algal population within 72 h exposure period gave the following results:

The NOE_rC (72h) and the NOE_yC (72h) were determined to be at least nominal 100 mg test item/L. The NOEC values might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The LOE_rC (72h), the LOE_yC (72h), the E_rC50 (72h) and the E_yC50 were clearly higher than nominal 100 mg test item/L (mean measured: clearly higher than 79 mg test item/L). No toxic effects against algae were observed at the limit of water solubility under these exposure conditions.