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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyltris(dimethylaminato)phosphorus(1+) tetrafluoroborate(1-)
EC Number:
EC Name:
Benzyltris(dimethylaminato)phosphorus(1+) tetrafluoroborate(1-)
Cas Number:
Molecular formula:
benzyltris(dimethylaminato)phosphorus(1+) tetrafluoroborate(1-)
Test material form:
solid: crystalline

Study design

Oxygen conditions:
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht was used as inoculum with a concentration corresponding
to 30 mg dry solids per litre. The treatment plant clarifies predominantly domestic wastewater of the region of Freiburg and has a capacity of
600.000 inhabitant equivalents. Sampling date of activated sludge was 20 September 2016. Dry solid of the activated sludge was determined
as 3.6 g/L by weight measurements after 3 h drying at 105°C (mean of triplicate measurements). The activated sludge was washed twice
with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Composition of medium:

A: Potassium dihydrogenphosphate KH2PO4 8.50 g
Dipotassium hydrogenphosphate K2HPO4 21.75 g
Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40 g
Ammonium chloride NH4Cl 0.50 g
are dissolved in demineralised water and made up to 1 litre.
B: Calcium chloride dihydrate CaCl2*2H2O 36.4 g
is dissolved in demineralised water and made up to 1 litre.
C: Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.5 g
is dissolved in demineralised water and made up to 1 litre.
D: Iron (III) chloride hexahydrate FeCl3 * 6H2O 0.25 g
is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1 litre.
For preparation of the mineral medium 10 mL of solution (A) is mixed with 800 mL demineralised water,
1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre.
Before use, the mineral medium is aerated for about one hour and the pH was measured.

Test temperature: 21.9 – 22.4°C (+/-1°C)
pH: 7.5-7.6
pH adjusted: no

The Oxitop-System from WTW, Weilheim was used as test system. It consists of narrownecked glass bottles with rubber sleeve inserts for NaOH pellets, in which the carbon dioxide evolved is absorbed. Magnetic stirrers are introduced into the bottles which are positioned on a stirrer platform. In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound and one reactor containing the test item and the reference item (toxicity control) were set up. The liquid volume was fixed as 164 mL each. The bottles were sealed tightly with the measuring heads with a measuring range of 500 – 1350 hPa. Every 112 minutes the current pressure was
measured and stored by each measuring head. At the end of the experiment the pressure data were read out via an infrared interface to the controller unit and afterwards the data were transferred via a RS232 interface using the Achat OC software of WTW to an Excel-file
where further data processing was carried out. Additionally several measured values were randomly read out and recorded by hand and were afterwards compared with the printed excel table for quality control.

164 mL of the dilution water with a pH of 7.6 were filled into the blank flasks. The test item was added into the test vessels via a stock solution. Afterwards 164 ml of the mineral medium was added to the test vessels. The stock solution of sodium acetate with a pH of 7.6 was added into the reference flasks and the stock solution of test item and sodium acetate was added to the toxicity control. After tempering the flasks to the incubation temperature for about 2 hours, 1.36 ml of the inoculum were added into each flask, one sodium hydroxide pellet was added to each rubber quiver inserted in the flasks and the flasks were sealed tightly with the measuring heads and the test was started. After 28 days the data were read
out and the pH in the flasks was measured.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Key result
% degradation (TOC removal)
Sampling time:
28 d

Any other information on results incl. tables

Test item:

The biodegradation of the test item was -1.9% within 28 days (mean of three replicates, see table 2).

Therefore the test item did not reach the criteria for ready biodegradability (60% ThOD and 10-d window).

The pH in the test bottles at the end of the test was 7.5 - 7.6.

Reference item:

The reference compound sodium acetate reached the pass level for ready biodegradability (60% ThOD and

10-day window) within 8 days (see table 2). The pH in the test bottles at the end of the test was 8.9 – 9.1.

Toxicity control:

The degradation in the toxicity control reached 36.1% within 4 days (see table 2) and was thus above the

criterion for inhibition effects to the inoculum (<25% on day 14). The test item had no toxic effect to the

inoculum according to the validity criteria of OECD 301. The pH in the vessel was 8.6.


The oxygen consumption of the blanks was 27.2 mg/L in 28 days (mean of three replicates, see table 1).

The pH in the vessels was 7.5.


The temperature range was 21.9 – 22.4 °C throughout the whole study.

Criteria met for the validity of the study:

The oxygen uptake of the inoculum blank was below 60 mg O2/L within 28 days. The pH value in the test and blank bottles was 7.5 – 7.6 at the end of the test. The difference of extremes of replicate values was less than 20%. The biodegradation of the reference item reached the pass level of 60% ThOD by day 4. The degradation extent in the toxicity control was above 25% in 14 days based on ThOD.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The degradation extent of the test item at the end of the test was -1.9% (mean of three replicates). The criterion for ready biodegradation was not met (degradation 60% and 10-dwindow), therefore the test item is not ready biodegradable.

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