Phosphotungstic acid

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:

2017-03-22 to 2017-04-21

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:

2010

Deviations:

no

Principles of method if other than guideline:

There were no deviations from the guideline.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Batch no: 160306
- Purity: > 99 %

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: direct addition by initial weight into the test flasks; test substance was weighed in the required amounts for the test concentrations directly to the test vessels
- Controls: negative control, procedure control
- Evidence of undissolved material: no
- Replicates:
6 replicates for the negative control,
3 replicates for each test substance concentration,
2 replicates for each reference substance concentration

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: wastewater treatment plant of Mannheim, Germany; collected on 22 Mar 2017; arrival in the test facility: 22 Mar 2017
- Preparation of inoculum for exposure:
After arrival of the activated sludge suspension in the test facility the suspension was sieved
with a fine woven mesh (mesh size about 1 mm). This suspension was pre-aerated over
night at room temperature. At the next day the sludge suspension was washed once with
drinking water and the suspension was adjusted to 3 g/L Dw.
- Initial biomass in the test vessel: 1.5 g/L Dw

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

No

Test temperature:

Test item (after incubation): 21.0 – 21.8 °C

pH:

Test item: 7.7-8.0

Nominal and measured concentrations:

Nominal: 1000, 500, 250, 125, 62.5 mg/L based on test substance without correction of purity and blank controls

Details on test conditions:

- Incubation time: 3 hours
- Test temperature: 21.0 – 21.8 °C
- Test vessels: glas-beakers (nominal volume 1L)
- Test volume: 500 mL
- Synthetic medium: 16 mL/test vessel of 100-fold concentrated OECD medium
- Oxygen concentration during aeration: > 2 mg/L
- Oxygen concentration immediately before measurement: > 7 mg/L
- Duration of the measurement of oxygen consumption: about 8 - 10 min (one replicate 10.1 min)
- Sludge concentration in the test vessel: 1.5 g/L Dw

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 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.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption: no

VALIDITY
The coefficient of variation of the six replicates of blank control was 5.4 % O2 consumption.
The EC50 of 3,5-dichlorophenol was 7.4 mg/L
The mean oxygen uptake of the blank controls was 21 mg/g*h
The test conformed to all validity criteria and is valid.

Results with reference substance (positive control):

The EC50 of the reference substance 3,5-dichlorophenol was in the range of 2-25 mg/L in 3 hours.

Reported statistics and error estimates:

The consumption rates were used for the determination of the ECx for the reference substance by the probit method based on Finney with the software TOXRAT Professional 2.10. The effect concentrations were given with an accuracy of 2 significant digits.

Table: Measured data of oxygen content of the test assays at the start and end of the evaluation time

Assay identification	BC1	BC2	BC3	BC4	BC5	BC6	RS1	RS2	RS3	RS4	RS5	RS6
O2 conc. Start [mg/L]	7.2	6.5	6.8	6.9	6.8	8.0	7.1	7.7	8.1	8.7	9.2	9.2
O2 conc. End [mg/L]	4.3	3.5	3.5	3.6	3.4	4.8	4.2	4.8	6.7	7.3	9.0	9.0
O2 consumption rate [mg/L]	2.9	3.0	3.3	3.3	3.4	3.2	2.9	2.9	1.4	1.4	0.2	0.2
O2 concumption rate in minutes	6.0	6.0	6.5	6.0	6.1	6.2	6.0	6.5	6.3	6.5	5.0	5.0
O2 consumption rate [mg/L×h], Rτ	29	30	30	33	33	31	29	27	13	13	2	2
Specific O2 consumption rate [mg/g×h]	19	20	20	22	22	21	19	18	9	9	1	1
Calculation of inhibition respiration [%], Iτ	-						6	13	58	58	94	94

Legend: RS = reference substance, BC = blank control

The respiration rate was calculated in mg O2/L×h. The oxygen consumption was measured over a period of 8-10 minutes. 5 - 6.5 minutes from the plotted track within a linear range of the graph was used for the determination of the respiration rate.

Validity criteria fulfilled:

yes

Description of key information

The 3-hour EC10 and EC50, respectively, of the test item was determined to be > 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The tocxicity to aquatic microorganisms was assessed in a Activated Sludge Respiration Inhibition Test in compliance with GLP criteria and OECD Guideline 209. Activated sludge from a municipal wastewater treatment plant of Mannheim (Germany) was exposed for 3 hours to test item concentrations of 1000, 500, 250, 125, 62.5 mg/L (nominal, based on test substance without correction of purity and blank controls). The test substance was added in the required amounts according to the test concentrations directly to the test vessels (glass beakers, volume 1L) with 234 mL deionized water. After addition of 250 mL of inoculum suspension (3 g/L DW) the incubation was started by aeration of the test vessels with pressured air. The vessels for the blank control assays were prepared to the same procedure without addition of test- or reference substance. 6 replicates for the blank control, 3 replicates for each test substance concentration and 2 replicates for each reference substance concentration were prepared. No abiotic control was tested. After 3 hours incubation at 20 ± 2°C the mixtures in the test vessels were placed subsequently for oxygen measuring. The oxygen uptake was measured for a period of about 8 to 10 minutes (one replicate: 10.1 min). The oxygen concentration during aeration was > 2 mg/L and immediately before measurement > 7 mg/L. In result, the 3-hour EC10 and EC50 of the test item was determined to be both > 1000 mg/L. In conclusion, no monotone concentration response exists because no inhibition was determined. Therefore the ECx calculation is not meaningful and the EC10 value is assumed to be greater than 1000 mg/L. The test conformed to all validity criteria and is valid, since the coefficient of variation of the six replicates of blank control was 5.4 % O2 consumption, the EC50 of the reference substance 3,5-dichlorophenol was 7.4 mg/L and the mean oxygen uptake of the blank controls was 21 mg/g*h.