Diphosphoric acid, compd. with piperazine (1:1)

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:

27 May 2003

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

Version / remarks:

April 1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Stock solution was prepared by adding 1.00 g of test substance to 500 ml dilution water. After a few seconds of ultrasonic treatment, the solution was clear. The pH was measured to be 4.0.
A stock solution of the reference substance was prepared according to OECD Guideline No. 209 by dissolving 0.1246 g of 3,5-dichlorophenol in 3 ml 2M NaOH solution, adjusting its pH to 8.0 with 2M HCl solution, and then filling up to 250 ml with ultrapure water.

- Controls: test medium without test item
- Test concentration separation factor: 3.2

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Approximately 10 litres of activated sludge were taken from an oxidation ditch situated at the district of Hazerswoude Dorp, the Netherlands on 25 May 2003. The oxidation ditch is used to treat domestic sewage.

- Pre-treatment: The sample was centrifuged and the supernatant discarded. The activated sludge was washed three times by centrifugation (10 min at 2000 rpm = max. 1218 g) and resuspensed in tap water twice and twice in dilution water.
The dry weight of the sludge suspension was determined by drying using an infrared drier. The mixed liquor suspended solids (MLSS) content was found to be 7.1 g/l. The suspension was aerated vigorously, and kept at approximately 20°C in the dark. The pH of the sludge suspension was measured to be 6.9 before use.
Fifty ml of synthetic sewage feed was added daily per litre of sludge suspension. Before use the sludge suspension was diluted with dilution water to approximately a MLSS content of 4 g/l. The dry weight was checked in the test and resulted in 4.5 g/l mixed liquor suspended solids.

- Initial biomass concentration: 4.5 g/l

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not specified in the study report.

Test temperature:

20°C

pH:

5.6 - 7.8

Dissolved oxygen:

Not applicable - respiration rate study

Nominal and measured concentrations:

The nominal test substance concentrations tested were: 0, 10, 32, 100, 320 and 1000 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: one litre glass beakers
- Aeration: yes, vigorously
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 4.5 g/l
- Preparation of test and control mixtures: Control mixtures of 16 ml synthetic sewage feed and 284 ml dilution water. Test mixtures were prepared by combining 16 ml synthetic sewage feed with the appropriate volumes of the test substance stock solution (2.5, 8.0, 25, 80 and 250 ml, respectively) and dilution water to a volume of 300 ml.

TEST MEDIUM/WATER PARAMETERS
- Source/preparation of dilution water: The (BOD) dilution water was prepared according to NEN Standard No. 6634 with ultrapure water.

OTHER TEST CONDITIONS
Adjustment of pH: no

INCUBATION
The incubation period was started at intervals of 10.5 minutes when the 200 ml of sludge suspension was added to the 300 ml of test or control mixture. The first and last of the various samples being incubated were controls without the test substance. All suspensions were incubated in a climate test chamber at approximately 20°C for exactly three hours.

EFFECT PARAMETERS MEASURED
Oxygen measurements: After three hours of incubation, a sample of each mixture was poured into a conical test flask (volume about 200 ml) and stirred vigorously on a magnetic stirrer. An oxygen electrode (Handy Delta OxyGuard®) was inserted into the sample and the oxygen concentration in the sample was continuously measured. The oxygen concentration was recorded every 1.5 minute during a period of about 10-11 minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range-finding study: no

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

487 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% confidence interval of 315-752 mg/l

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The dry weight determination of the sludge suspension used in the final test resulted in a mixed liquor suspended solids content of 4.5 g/l.
The oxygen measurements in the mixtures containing the concentrations of the test substance are shown in attachment “T-1063 – results respiration of activated sludge.pdf”. The calculated respiration rates, their correlation coefficients and the percentages inhibition of the respiration rate are shown in “T-1063 – results respiration of activated sludge.pdf”.

At the higher concentration the pH of the test medium is less than 6.0. This might have influenced the respiration rate. Based on the results this influence is considered negligible compared with the toxic effects.

The maximum inhibition was found to be 78.4 % at a nominal test substance concentration of 1000 mg/l.
The EC20 value was calculated to be 217 mg/l with a 95% confidence interval of 167 - 281 mg/l.
The EC80 value was calculated to be > 1000 mg/l extrapolated 1090 mg/l with a 95% confidence interval of 729 - 1640 mg/l.

Results with reference substance (positive control):

The oxygen measurements in the mixtures containing the three concentrations of the reference substance are shown in attachment “T-1063 – results respiration of activated sludge.pdf”. The calculated respiration rates, their correlation coefficients and the percentages inhibition of the respiration rate are shown in “T-1063 – results respiration of activated sludge.pdf”. The EC50 of 3,5-dichlorophenol could not be calculated because no proper relationship between the adverse effect and the test substance concentration was found. However, the EC50 was estimated to be > 5 mg/l and < 30 mg/l, which is within the range prescribed by OECD Guideline No. 209.

Validity criteria fulfilled:

yes

Conclusions:

The EC20, EC50 and EC80 values for T-1063 FM were 217, 487 and >1000 mg/l, respectively. The NOEC was assessed to be 100 mg/l

Description of key information

EC₅₀ (3h): 487 mg/l, NOEC(3h): 100 mg/l (OECD guideline 209)

Key value for chemical safety assessment

EC50 for microorganisms:

487 mg/L

EC10 or NOEC for microorganisms:

100 mg/L