Amidinourea phosphate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2017 to 27 Sept 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16VL8189
- Expiration date of the lot/batch:03 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (=< 30 deg C), dark, dry

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Activated sludge, municipal wastewater treatment plant Breisgauer Bucht, 30 mg dry solids per litre
Sampling date of activated sludge was on 09 May 2017. The dry solid content of the activated sludge was 4.8 g/L. It was determined by weight measurements after drying at 105°C for 3 hours (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):

29 d

Initial conc.:

10 g/L

Based on:

test mat.

Remarks:

corresponds to 20.0 mg/L organic carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- 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. The pH is checked and, if necessary, adjusted to 7.4.
B: Calcium chloride dihydrate CaCl2 * 2H2O 36.40 g is dissolved in demineralised water and made up to 1 litre.
C: Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.50 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 900 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre.
- Additional substrate: No
- Solubilising agent (type and concentration if used): Deionised water (DOC concentration of the deionised water used for the test was 0.37 mg/L)
- Test temperature: 21.1 - 23.6 deg C
- pH: 7.4
- pH adjusted: no
- Aeration of dilution water: No
- Suspended solids concentration: dry solid content of the activated sludge was 4.8 g/L
- Continuous darkness: no, diffuse light

TEST SYSTEM
- Culturing apparatus: 1000 mL gas wash bottles with Teflon-sealing
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: aeration with CO2 free air
- Measuring equipment: ICmeasurement with total carbon analyser
- Test performed in closed vessels due to significant volatility of test substance: No
- Details of trap for CO2 and volatile organics if used:
The CO2-free air production system consists of an air compressor, three 1000 mL gas wash bottles filled with dry soda lime in series followed by one bottle filled with 0.05 M NaOH (sodium hydroxide). At the end of the system is one gas wash bottle filled with demineralised water, followed by an empty one to catch any drops of condensation water. A colour change of the soda lime from white to blue indicates that the CO2 absorption capacity is depleted. The CO2-free air is passed on to an air distributor with two input and 22 output channels and through PE-tubes. Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septa were used as reactors. The liquid volume was fixed as 1500 mL each. Mixing was performed by magnetic stirrers with 2 cm stir bars. The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series each filled with 200 mL 0.2 M NaOH. Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 mL PE syringes.

SAMPLING
- Sampling frequency: Day 1 and thereafter, 4th, 7th, 11th, 15th, 21st and 28th day
- Sampling method: 4 mL NaOH from the first of two CO2 absorber flasks connected in line was sampled and the IC's were determined
- Sample storage before analysis: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: No.
- Toxicity control: 24.8 mL of the stock solution with the test item (10.0 g/L) and 5.15 mL of the reference stock solution (10.0 g/L) were added into the toxicity control vessel. This corresponds to a concentration of 40.0 mg/L organic carbon.
- Other: Reference substance: 10.0 g/L sodium benzoate in water was prepared. 5.15 mL of this stock solution were added into the reference vessels, corresponding to a concentration of 20.0 mg/L organic carbon.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Not performed

Test performance:

Toxicity control: The degradation extent in the toxicity control reached a value >25% within 4 days, the test substance had no inhibitory effect on the inoculum.
Reference item: The reference compound sodium benzoate reached the pass levels for ready biodegradability within 4 days
Blank:The mean CO2-evolution of the blank flasks was 35.9 mg/L on day 28 after acidification

Test parameters
Before adding the test item, the IC in the reactor was determined, but only insignificant amounts of IC (0.15 mg/L) were found.
The IC-concentration of the NaOH in the second CO2-absorber flasks in line, used as protective flasks, was below 7 ppm and was not considered in the data processing, because CO2 absorption from room air was its source.  The temperature was 21.1 – 23.6°C throughout the whole study.
The aeration rate was in the tolerated range of 1.6 – 5.5 bubbles/second (counted bubbles: 2.1 – 4.6 bubbles/second)

Criteria of validity
The IC content in the test vessel was 0.15 mg/L and therefore less than 5% of the TOC introduced with the test item.
The CO2 evolution in the inoculum blank at the end of the test was 35.9 mg/L.
The difference of extremes of replicate values of the test item at the end of the test was 11.6%.
The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 4.
The degradation extent in the toxicity control reached a value >25% by day 4 (38.5% ThCO2). .

Key result

Parameter:

% degradation (CO2 evolution)

Value:

-3.6

Sampling time:

28 d

Details on results:

No degradation of the test item could be observed during the test duration. The mean degradation extent at the end of the test was -3.6% (28 d after acidification, mean of three replicates)

Results with reference substance:

The reference compound sodium benzoate reached the pass levels for ready biodegradability within 4 days

Reactor	Day	0	4	7	11	15	21	28	29
7	Test flasks	0	1.5	1.1	0.3	-1.7	-1.4	2.8	2.3
8		0	1.5	0.6	-1.1	-3.2	-4.4	-2.9	-3.9
9		0	-1.1	-1.8	-3.6	-5.7	-7.7	-7.8	-9.3
4	Reference	0	68.1	81.5	86.9	90.0	91.8	90.1	87.7
5		0	73.2	80.9	86.2	94.4	91.4	92.9	87.9
6		0	66.0	84.3	91.7	96.9	999.0	101.4	99.7
10	Toxicity control	0	38.5	41.0	41.4	41.0	42.2	44.0	41.8
Mean (1-3)	Blanks	0	9.9	13.1	17.3	21.0	26.6	33.3	35.9

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

No degradation of the test item could be observed during the test duration. The mean degradation at the end of the test (28 d after acidification) was -3.6% (mean of three replicates). The test item did not reach the criteria for ready biodegradability (60% of ThCO2 within a 10-d window). The degradation of the toxicity control after 14 days was >25%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.

Executive summary:

A ready biodegradation test in accordance with OECD Guideline 301B (CO2 evolution) was performed.

No degradation of the test item could be observed during the test duration. The mean degradation at the end of the test (28 d after acidification) was -3.6% (mean of three replicates). The test item did not reach the criteria for ready biodegradability (60% of ThCO2 within a 10-d window). The degradation of the toxicity control after 14 days was >25%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.

Description of key information

No degradation of the test item could be observed during the test duration. The mean degradation at the end of the test (28 d after acidification) was -3.6% (mean of three replicates). The test item did not reach the criteria for ready biodegradability (60% of ThCO2 within a 10-d window). The degradation of the toxicity control after 14 days was >25%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater