2-ethylhexyl 10-ethyl-4,4-dimethyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test mixtures were prepared at 15 minute intervals starting with the first control. The control contained 16.0 mL of synthetic sewage, 200 mL of inoculum, and enough municipal water to bring the total volume up to 500 mL. The mixture was promptly aerated at a rate sufficient to provide aerobic conditions and mixed using stir plate and magnetic stir bar to keep the solids in suspension. Subsequent mixtures contained 16.0 mL of synthetic sewage, 200 mL of inoculum, the appropriate amount of test substance or reference substance stock solution, and enough municipal water to bring the total volume up to 500 mL. A second control was prepared. All mixtures were aerated and stirred for three hours.
- Eluate: NA
- Differential loading: NA
- Controls: Control and reference substance control – 3,5-dichlorophenol was prepared by dissolving 500 mg of the reference substance in 10 mL of 1N NaOH and then diluting to 20 mL with NANO®pure water. The NANO®pure water is not known to contain any contaminants that are reasonably expected to be present at levels capable of interfering with the study. While stirring, 9.2 mL of 1N H2SO4 was added to reach the point of incipient precipitation. The solution of 3,5-dichlorophenol was then diluted to a final volume of 1L with NANO®pure water. The reference substance solution was administered to the reference group by volumetric addition. Volumetric addition is the most appropriate route of administration of aqueous solutions.- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: No
- Method of cultivation: Activated sludge collected for the Cambridge Waste water Treatment Plant, Cambridge, Maryland on August 03, 2009 was utilized as the inoculum for the test. The Cambridge facility receives wastes from predominantly domestic sources. The sludge was sieved using a 2 mm screen and allowed to settle for approximately 30 minutes. The supernatant above the settled solids was removed and the total suspended solids (TSS) concentration of the settled sludge was determined. Total suspended solids in the settled sludge were adjusted to a nominal concentration of approximately 4000 mg/L by dilution with municipal water. 50 L of synthetic sewage was added to each liter of adjusted sludge. The sludge was maintained at a temperature of 21 ± 1 ◦C and continuously aerated overnight. On the day if use in the study
- Preparation of inoculum for exposure: The biological test system was a consortium of microorganisms common to the activated sludge treatment process. The organisms responsible for the decomposition of organic materials are principally aerobic and facultative aerobic bacteria. The test system was chosen because it is a representative of a treatment process that may receive the test substance.
- Pretreatment:
- Initial biomass concentration: No data

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

not applicable

Hardness:

no data

Test temperature:

20.1- 21.2◦C.

pH:

7.7

Dissolved oxygen:

6.8 to 0.6

Salinity:

no data

Nominal and measured concentrations:

10, 30 100, 300, and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flasks the transferred to fill 300 mL biochemical Oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements.
- Type (delete if not applicable): 1000 mL Erlenmeyer flasks
- Material, size, headspace, fill volume: 1000 mL
- Aeration: Yes, 3 hours
- Type of flow-through (e.g. peristaltic or proportional diluter): NA
- Renewal rate of test solution (frequency/flow rate): NA
- No. of organisms per vessel: NA
- No. of vessels per concentration (replicates): 2-3
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 3
- Biomass loading rate: NA


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: NANO®pure water
- Total organic carbon: No data
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: No
- Intervals of water quality measurement: No data


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): dissolved oxygen concentrations were measured every 10 seconds over a 15 minute period. Respiration rates and percent inhibitions were calculated from this.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Justification for using fewer concentrations than requested by guideline: NA
- Range finding study
- Test concentrations: NA
- Results used to determine the conditions for the definitive study: NA

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Treatment/Nominal Concentration Respiration Rate Percent Inhibition
MgO2/L/hour
Control 1 36.0 NA
Control 2 41.1 NA

3,5-dichlorophenol 3 mg/L 36.0 6.7
3,5-dichlorophenol 5 mg/L 23.2 39.9
3,5-dichloropheno50 mg/L 6.7 82.6

DMT(EHTG) 10 mg/L 48.5 -25.6
DMT(EHTG) 30 mg/L 46.5 -20.4
DMT(EHTG) 100 mg/L 102.9 -166.7
DMT(EHTG) 300 mg/L 39.4 -2.1
DMT(EHTG) 1000 mg/L 38.6 0.0


NA – Not Applicable

Results with reference substance (positive control):

The EC50 (3 hours) of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L.
- Results with reference substance valid? Yes
- Relevant effect levels: The EC50 value for DMT(EHTG) in this study is greater that 1000 mg/L, the highest concentration tested.

Reported statistics and error estimates:

When the dose response pattern (percent inhibition versus test substance concentration) allowed for the calculation of an EC50 value, the data were analyzed using the computer program of C.E. Stephan. The program was designed to calculate the EC50 value and the 95% confidence interval by probit analysis, the moving average, or binomial probability with nonlinear interpolation.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value for the test substance in this study is greater than 1000 mg/L which was the highest concentration tested.

Executive summary:

The effect of the test substance on activated sludge microorganisms maintained in an aerobic environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209). The test contained control, reference and treatment groups. The control replicates were used to determine the background respiration rate of the sludge and were not dosed with the test or reference substance. The reference group was dosed with 3,5 -dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50 mg/L. The treatment group was dosed with the test substance at concentrations of 10, 30, 100, 300 and 1000 mg/L. After an exposure period of three hours, the respiration rates of the test solutions were measured using a YSI Model 50B-115 Dissolved Oxygen Meter. The respiration rates in the two controls were 36.0 and 41.1 mg O2/L/hr. The difference between the two control respiration rates was approximately 13%, and was within the 15% difference limit established for the test. The validity of the test was further supported by the results from the 3,5-dichlorophenol reference group, which resulted in an EC50 of 19.5 mg/L. The EC50 for the reference substance was within the 5 to 30 mg/L range considered acceptable for the test. The highest percent inhibition value obtained in the study, for the test substance vessels, was 0.0%. The EC50 value for the test substance is therefore greater than 1000 mg/L, the highest concentration tested.

Description of key information

EC50 (3-hr) >1000 mg/L for activated sludge in a respiration inhibition test (OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The effect of the test substance on activated sludge microorganisms maintained in an aerobic environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209). The test contained control, reference and treatment groups. The control replicates were used to determine the background respiration rate of the sludge and were not dosed with the test or reference substance. The reference group was dosed with 3,5 -dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50 mg/L. The treatment group was dosed with the test substance at concentrations of 10, 30, 100, 300 and 1000 mg/L. After an exposure period of three hours, the respiration rates of the test solutions were measured using a YSI Model 50B-115 Dissolved Oxygen Meter. The respiration rates in the two controls were 36.0 and 41.1 mg O2/L/hr. The difference between the two control respiration rates was approximately 13%, and was within the 15% difference limit established for the test. The validity of the test was further supported by the results from the 3,5-dichlorophenol reference group, which resulted in an EC50 of 19.5 mg/L. The EC50 for the reference substance was within the 5 to 30 mg/L range considered acceptable for the test. The highest percent inhibition value obtained in the study, for the test substance vessels, was 0.0%. The EC50 value for the test substance is therefore greater than 1000 mg/L, the highest concentration tested.