Ammonium 7-(2,6-dimethyl-8-(2,2-dimethylbutyryloxy)-1,2,6,7,8,8a-hexahydro-1-naphthyl)-3,5-dihydroxyheptanoate

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:

29th October 2010 to 23rd December 2010

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Source: Axenic, uni-cellular, liquid slope cultures of algae were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine
Laboratory, Dunbeg, Oban, Argyll, Scotland and arrived on 3 November 2010.
- Age of inoculum (at test initiation): an inoculum in the log phase of growth, characterised by a cell density of 7.30 x 105 cells/mL.
- Method of cultivation: Sterile algal nutrient medium inoculated with cells aseptically removed from the slope culture

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: Sterile algal nutrient medium
- Any deformed or abnormal cells observed: Noted during test

Test type:

static

Water media type:

other: Filtered, dechlorinated tap water

Total exposure duration:

72 h

Test temperature:

21 to 24°C

Nominal and measured concentrations:

The definitive test concentrations, selected based on the results of the range finding test, were
0.954, 3.05, 9.77, 31.3 and 100 mg/L.

Details on test conditions:

The method of preparation used during the definitive test was based on the results of a range finding test. The test substance (100 mg) was dissolved in culture medium (1 L) in a volumetric flask. The contents of the flask were shaken vigorously and treated by ultrasound for five minutes before it was either used directly at the highest test concentration or further diluted to provide the test media at the lower concentrations.
An aliquot (6.85 mL) of the secondary algal inoculum was added to a portion (500 mL) of the test medium at each concentration, to give an initial cell density of 1 x 104 cells/mL. An aliquot (100 mL) of the appropriate inoculated test medium was added to each of the test vessels.
Six flasks were established and incubated for the control group and three flasks for each test group, plus an additional flask at nominal concentrations of 0.954 and 100 mg/L, which contained test medium but no algal cells. The media remaining in the preparation flasks were used for water quality measurements at the start.
Before the start of the test, the required number of empty test vessels (250 mL conical flasks), were loosely stoppered with foam bungs, covered with aluminium foil that was secured by autoclave tape and sterilised by autoclaving (121°C for at least 15 minutes). After the addition of the inoculated test medium (100 mL), each flask was then loosely plugged with a foam bung.
The control cultures were prepared as for the test medium except that no test substance was added and a larger volume (800 mL) of medium was made.
The definitive test concentrations, selected based on the results of the range finding test, were 0.954, 3.05, 9.77, 31.3 and 100 mg/L.
Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities measured using a Coulter Z Series Particle Count and Size Analyser. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts of uninoculated dilution media. The presence of any abnormal cells was also noted during screening of each test level.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.45 mg/L

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to Simvastatin Ammonium Salt, the EbC50, ErC50 and EyC50
respectively were 32.3, 120 and 27.4 mg/L.
The “no observed effect concentration” (NOEC) for area under the growth curve, growth rate
and yield was 2.45 mg/L.

Executive summary:

The effect of Simvastatin Ammonium Salt on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions.
The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex IV to Commission Regulation (EC) No 761/2009 (O.J. No. L220/36, 2009) Part C, Method 3 “Freshwater Algae and Cyanobacteria, Growth Inhibition Test” and Procedure 201of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (2006).
Triplicate algal cultures, with an initial cell density of 1 x 104 /mL, were exposed to Simvastatin Ammonium Salt at nominal concentrations of 0.954, 3.05, 9.77, 31.3, and 100 mg/L. The test media were prepared in OECD algal medium from an aqueous stock solution, which was made by the direct addition of the test substance to the dilution medium. To aid dissolution, the aqueous stock solution was treated with ultrasound for five minutes. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 21.9 to 23.3°C for 72 hours.
The measured levels of Simvastatin Ammonium Salt in samples of the test cultures ranged between 86 and 102% of their nominal values at the start of the test and between 75 and 92% of their nominal values after 72 hours; between 88 and 97% of their starting values. The overall mean measured levels of Simvastatin Ammonium Salt were 0.847, 2.45, 8.74, 29.1, and 95.7 mg/L.
Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve, growth rate and yield. The following values were derived from the data:
Area under the growth curve E_bC₅₀ (72 h) : 32.3 mg/L (95% confidence limits, 29.2 & 35.8 mg/L)
Average specific growth rate ErC₅₀ (0 - 72 h) : 120 mg/L (95% confidence limits, 112 & 126 mg/L)
Yield E_yC₅₀ (0 - 72 h) : 27.4 mg/L (95% confidence limits, 24.2 & 31.2 mg/L)
No observed effect concentration (NOEC) : 2.45 mg/L 

Key value for chemical safety assessment

EC50 for freshwater algae:

120 mg/L

EC10 or NOEC for freshwater algae:

2.45 mg/L