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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:070602
- Purity: 92.7% w/w
- Carbon content: 27%
- Expiration date: 6 June 2019
- Purity test date: 31 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item is very hygroscopic, it should be stored in the closed original container.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item is used via stock solution.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge, predominantly municipal wastewater treatment plant AZV Staufener Bucht, 30 mg dry solids per litre
- Storage length: 2 days
- Preparation of inoculum for exposure: washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.

- Concentration of sludge: 9.6 ml in 1500 ml of mineral medium corresponding to 30 mg/l dry solids
- Initial cell/biomass concentration: initial dry solid content: 4.7 g/l (mean of 3 measurements)
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 g/L
Based on:
TOC
Parameter followed for biodegradation estimation
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.
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 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:
CO2-absorption medium: 64.09 g NaOH was dissolved in 8000 mL deionised water in closed vessels (0.2 M NaOH). The inorganic carbon concentration of the 0.2 M NaOH was determined (IC = 3.229 mg/L).
- Solubilising agent (type and concentration if used): water
- Test temperature: 20.2 – 22.2°C
- Aeration rate: 1-100 ml/min (1.6 – 5.5 bubbles/s)
- Suspended solids concentration: activated sludge, 45 mg/1500 ml
- Continuous darkness: no diffuse light
- Other:

TEST SYSTEM
- Culturing apparatus: reactor (2000 ml) gas wash flasks with GL14 hole-caps and frit pipes
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: CO2-free air compressor and a raw of air-tubes (air distributor) with 2 input and 22 output channels, with a specific aeration rate, and through PE-tubes.
- Measuring equipment: inorganic carbon (IC) measurement with a total carbon analyser (TOC-L, Shimadzu) by purging the inorganic carbon with H3PO4 (25%) using a non-dispersive infrared (NDIR) detector.
- Test performed in closed vessels due to significant volatility of test substance: YES Gas wash bottle (2000 ml) with lateral connecting pieces for butyl rubber septa
- Details of trap for CO2 and volatile organics if used:
CO2 trapping for the CO2-free air production: three 1000 mL gas wash bottles filled with dry soda lime in series followed by one bottle filled with 0.1 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 color change of the soda lime from white to blue indicates that the CO2 absorption capacity is depleted.
CO2 trapping during experiments: 2 250 ml gas wash bottles in series each filled with 200ml 0.2 M NaOH.
- Other:

SAMPLING
- Sampling frequency: before the experimentation D0, D4, D7, D11, D14, D21 and D28
- Sampling method: through the butyl rubber septum with a 5 ml PE syringe
- Sample storage before analysis: immediately closed with sealing film

CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 reactors
- Abiotic control: 3 reactors with the reference compound
- Toxicity control: 1 reactor with the test system + the reference compound (corresponding to a concentration of 40 mg/L organic carbon)
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
None
Test performance:
The degradation extent in the toxicity control was 41.5% within 14 days. Hence, the test item had no inhibitory effect on the inoculum at the test concentration of 40 mg TOC/L
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
-5.9
St. dev.:
1.5
Sampling time:
28 d
Details on results:
One hypothesis for the negative result is a problem of CO2 absorption by the test item itself.

Any other information on results incl. tables

Ultimate biodegradation after X days [% of ThCO2]

 reactor  day  0  4  7  11  14  21  28  28 (incl. IC dissolved in reactor)
 11  Test  flasks  0  -2.6  -4.4  -5.9  -6.4  -7.5  -8.2  -8.0
 12     Test  flasks  0  -1.0  -1.9  -3.6  -4.0  -4.6  -4.8  -4.9
 13    Test  flasks  0  -0.7  -1.9  -3.1  -3.7  -4.0  -4.8  -4.8
    mean value test flasks  0.0  -1.4  -2.7  -4.2  -4.7  -5.4  -5.9  -5.9
    standard deviation  0.0  0.8  1.2  1.2  1.2  1.5  1.6  1.5
 4  reference flasks  0  70.6  83.7  84.8  86.7  87.3  90.3  90.9
 5   reference flasks  0  73.1  84.0  86.2  87.9  88.1  89.1  89.5
 6   reference flasks  0  75.6  86.9  87.7  88.8  90.1  90.0  91.0
    mean value refrence flasks  0.0  73.1  84.9  86.2  87.8  88.5  89.8  90.5
    standard deviation  0.0  2.0  1.5  1.2  0.8  1.2  0.5  0.7
 14  toxicity control test item reference item  0  35.8  39.3  40.7  41.6  43.0  42.5  42.6

Mean CO2 -evolution of blank flasks (1 -3) after x days:

 Day  0  4  7  11  14  21  28  28 (incl. IC dissolved in reactor)
 CO2 -evolution [mg/l]  0  7.5  10.3 13.2   14.8  18.6  22.1  23.4

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
There was no degradation of the test item observed during the test duration. The degradation extent of the test item at the end of the test was -5.6% (mean of three replicates, see table 1 and figure 1).

Therefore the test item did not reach the criteria for ready biodegradability (60% of ThCO2 within a 10-day window).

The degradation extent in the toxicity control was 41.5% within 14 days (see table 1). Hence, the test item had no inhibitory effect on the inoculum at the test concentration of 40 mg TOC/L.
Executive summary:

The biodegradability of “X330” was tested in the CO2 Evolution Test according to OECD 301 B (July 1992). The test item was added via stock solution (10 g/l in water) inoculated with activated sludge, municipal wastewater treatment plant AZV Staufener Bucht, 30 mg dry solids per litre during 28 days. Degradation is followed by determining the carbon dioxide produced and absorbed to sodium hydroxide via IC-measurement (IC = inorganic carbon). The amount of carbon dioxide produced from the test item less the amount derived from the blank inoculum is expressed as a percentage of ThCO2 (theoretical amount of CO2). The pass level for ready biodegradability is 60% of ThCO2 and must be reached within a 10-d window. The 10-d window begins when the degree of biodegradation reaches 10%.

There was no degradation of the test item observed during the test duration. The degradation extent of the test item at the end of the test was -5.6% (mean of three replicates, see table 1 and figure 1).

Therefore the test item did not reach the criteria for ready biodegradability (60% of ThCO2 within a 10-day window).

The degradation extent in the toxicity control was 41.5% within 14 days (see table 1). Hence, the test item had no inhibitory effect on the inoculum at the test concentration of 40 mg TOC/L.