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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th August 2010 to 20th August 2010

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

GLP compliance:

yes

Vehicle:

not specified

Details on test solutions:

A stock solution (2.5 g/L) of Simvastatin ammonium Salt was prepared in Reverse Osmosis (RO) water and aliquots added ti the respective berakers. Antifoam (silicone in water) was added to each beaker (100μl) to prevent foaming in mixture. Additions of synthetic sewage and microbial inoculum were made at 15 min intervals to give the final test concentrations as follows:

Simvastatin Ammonium Salt mg/L: 10, 30, 100, 300 and 1000. Control: 0
3,5-DCP mg/L: 3, 10 and 32

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Worlingworth Sewage Treatment Works, Suffolk, UK

- Preparation of inoculum for exposure: On the day of collection, an aliquot (10ml) of the activated sludge was filtered through a dried and pre-weighed Whatman GF/C filter paper. The filter was then dried again at approx 105 oC for at least 1 hour, allowed to cool in a desiccator, then re-weighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50ml) was added to the stock of activated sludge and this was aerated overnight.

- Initial biomass concentration: On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200ml) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.

Test type:

not specified

Water media type:

other: Reverse Osmosis Water

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Initial Temperatures (oC)
Control - 20.3
Test Mixture (mg/L):
10 - 20.3,
30 - 20.4,
100 - 20.6,
300 - 20.7,
1000 - 20.6,
1000 - 20.7,
1000 - 20.8.

Final Temperatures (oC)
Control - 20.4
Test Mixture (mg/L):
10 - 20.4,
30 - 20.3,
100 - 20.3,
300 - 20.3,
1000 - 20.2,
1000 - 20.1,
1000 - 20.1.

pH:

Start of test: Test mixture (mg/L):
10 - pH 7.5
30 - pH 7.4
100 - pH 7.4
300 - pH 7.3
1000 - pH 7.4
1000 - pH 7.4
1000 - pH 7.3
Control - pH 7.4

Completion of test: Test mixture (mg/L):
10 - pH 7.7
30 - pH 7.7
100 - pH 7.7
300 - pH 6.8
1000 - pH 7.1
1000 - pH 7.1
1000 - pH 7.0
Control - pH 7.8

Dissolved oxygen:

Start of test: Test mixture (mg/L):
10 - 3.7
30 - 3.8
100 - 4.5
300 - 5.5
1000 - 7.0
1000 - 7.0
1000 - 7.0
Control - 5.5

Completion of test: Test mixture (mg/L):
10 - 0.5
30 - 1.0
100 - 1.0
300 - 2.0
1000 - 4.5
1000 - 4.5
1000 - 4.0
Control - 1.5

Details on test conditions:

The prepared mixtures were aerated for 3 hours in a thermostatically-controlled water bath, using an aerator connected to a laboratory supply of oil-free compressed air (one litre/minute). Following the exposure period, a well mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle (nominal capacity 270mL).T
The rate of dissolved oxygen consumption was measured over a period of approximately 10 minutes or until the dissolved oxygen concentration fell below 2 mgO2/L using a Yellow Springs Instrument (YSI) dissolved oxygen meter with a self-stirring bottle probe connected to a chart recorder. The pH and temperature were measured at the start and end of the test.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

891 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

174 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The 3-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 10.9 mg/L (95% confidence limits, 7.7 TO 15.4 mg/L)

Validity criteria fulfilled:

yes

Conclusions:

Inhibition of respiration rates were observed at Simvastatin Ammonium Salt concentrations between 300 mg/L and 1000 mg/L, with the highest concentration (1000 mg/L) showing 52 to 53 % inhibition (mean 52%).

The three hour EC50 of Simvastatin Ammonium Salt was calculated to be 174 mg/L (95% confidence limits 62.1 to 359 mg/L) and the three hour NOEC of Simvastatin Ammoniun Salt was considered to be 100 mg/L.  

The three hour EC50 for Simvastatin Ammonium Salt was calculated to be 891 mg/L (95% confidence Limits, 613 to 1350 mg/L)

Executive summary:

The effect of Simvastatin Ammonium Salt on the respiration rate of activated sludge was assessed by the methods detailed in Directive EC 440/2008 Method C.11 'Biodegradation - Activated Sludge Respiration Inhibition Test' and OECD Test Guideline 209 'Activated Sludge Respiration Inhibition Test' (1984). The three hour No Observed Effect Concentration (NOEC) of the pharmaceutical test substance was defined as the highest tested nominal concentration of test substance lower than the calculated EC₂₀ showing no effect. 

Samples of activated sludge (suspended solids 1.6 g/L) fed with synthetic sewage, were exposed to dilutions of the test substance for three hours. Their rates of oxygen consumption were determined using an oxygen electrode and were compared with those of the controls that contained activated sludge and synthetic sewage alone and were established at the beginning and the end of the test. 

The study employed nominal Simvastatin Ammonium Salt concentrations of 10, 30, 100, 300 and (triplicate) 1000 mg/L. The reference inhibtor 3,5-dichlorophenol (3,5-DCP) was employed at 3, 10 and 32 mg/L, as a positive control. 

The specific respiration rate of the control culture was established at the end of the test (Control 2, 28.3 mgO₂/g/h) was 109% of the rate established at the start (Control 1, 25.9 mgO₂/g/h). The three hour 50% effect concentration (EC₅₀) for 3,5-DCP was calculated to be 10.9 mg/L (95% confidence limits, 7.7 to 15.4 mg/L). These results show that the sample of activated sludge employed was sensitive to inhibition. 

Inhibition of respiration rates were observed at Simvastatin Ammonium Salt concentrations between 300 mg/L and 1000 mg/L, with the highest concentration (1000 mg/L) showing 52 to 53 % inhibition (mean 52%).

The three hour EC20 of Simvastatin Ammonium Salt was calculated to be 174 mg/L (95% confidence limits 62.1 to 359 mg/L) and the three hour NOEC of Simvastatin Ammoniun Salt was considered to be 100 mg/L.  

The three hour EC₅₀ for Simvastatin Ammonium Salt was calculated to be 891 mg/L (95% confidence Limits, 613 to 1350 mg/L)

Key value for chemical safety assessment

EC50 for microorganisms:

891 mg/L

EC10 or NOEC for microorganisms:

100 mg/L