Bis[O,O-bis(2-ethylhexyl) dithiophosphorato-S,S']dioxodi-μ-thioxodimolybdenum

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

- Method:
The test item was coated on glass pearls (approximately 2 mm diameter) to enhance contact between test item and activated sludge. Acetone was used as solvent to prepare the stock solutions. All stock solutions were clear and had a slightly yellow to dark brown color. Color intensity increased with increasing concentration of the stock solutions. Approximately 30 g glass pearls were spiked with these stock solutions and mixed with 200 mL Milli-RO, after evaporation of the acetone. The test item – Milli-RO water mixtures were magnetically stirred for a short period.
The pH of the test dispersions was in the range 7.3-7.6.
- Controls: test medium without test item.
- Solvent/glass pearl controls: test medium without test item but treated in the same way as the test item solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Method of cultivation / Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. 250 ml sludge was dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.3 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: 3.0 g/L (dry weight)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

1.8 mM (180 mg CaCO3/L)

Test temperature:

20 - 22 °C

pH:

test start: 7.3 - 7.6 test end: 7.5 - 8.0

Dissolved oxygen:

>60% saturation (> 5 mg/L at 20°C)

Nominal and measured concentrations:

nominal: 1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass bottles/vessels
- Type (delete if not applicable): open
- Aeration: clean, oil-free air
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6
- No. of vessels per abiotic control (replicates): 1 (within the range finding study)
- Sludge concentration (weight of dry solids per volume): 1.5 g/L (dry weight)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands): CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L.
OTHER TEST CONDITIONS
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): dissolved oxygen concentration after 3 hours contact time, continuously recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L plus abiotic control at 1000 mg/L
- Results used to determine the conditions for the definitive study: 28%, 34% and 39% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. No oxygen uptake from abiotic processes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95% confidence interval: 33 – 121 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

8.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Remarks on result:

other:

Remarks:

95% confidence interval: 1.8 – 19.1 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Details on results:

- Blank controls oxygen uptake rate: 31 O2/g.h (range 28-35; SD 2.43, n=6)
- Coefficient of variation of oxygen uptake rate in control replicates: 8%

Results with reference substance (positive control):

Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h): 3.9 mg/L (95% confidence limit 1.3-6.7 mg/L); the result is within the guidelines-recommended range of 2–25 mg/L

Reported statistics and error estimates:

For the comparison of the respiration rate in the Control and Solvent/glass pearl control a student –t test for Homogeneous variances was performed.
ECx
For the reference item, calculation of the EC50 was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For the test item, calculation of the ECx was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.
The EC50 could not be calculated because effects were below 50%.
NOEC determination
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the pooled control and solvent/glass pearl control revealed significant inhibition of the respiration rate (Williams Multiple Sequential t- test Procedure, α=0.05, one-sided, smaller).

Calculations were performed using ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

There was no statistically significant difference in the respiration rate between the control and the solvent/glass pearl control. Therefore, the controls were pooled for further statistical calculations.

No statistically significant inhibition of the respiration rate of the sludge was recorded at or below 10 mg per litre. The three highest concentrations showed a similar significant inhibitory effect on the aerobic waste water (activated sludge) bacteria (see attached document "S-930 - Respiration Rates and Inhibition.pdf"). This effect might be caused by the fact that concentrations were tested resulting in comparable bioavailability of the test item for the bacterial population, despite all efforts to ensure a good homogeneity (coated on glass pearls, stirring/aeration). The bioavailability could be the rate limiting factor above a certain threshold.

Validity criteria fulfilled:

yes

Remarks:

The specific respiration rates in the controls were ≥20 mg O2/g/h and the coefficient of variation was ≤ 30 %

Description of key information

Toxicity to microorganisms: not toxic to microorganisms: EC50 > 1000 mg/L, EC10, NOEC 8 and 10 mg/L (OECD 209)

Additional information

EC50 for microorganisms > 1000 mg/L

EC10 for microorgansims 8 mg/L

NOEC for microorgansims 10 mg/L