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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 2017 to 31 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
July, 1992
Deviations:
yes
Remarks:
The temperature during the test was 19.2 - 22.3°C and therefore temporarily below the range required by the guideline (22 ± 2°C).
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly 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 and has a capacity of 600 000 inhabitant equivalents. Sampling date of activated sludge was on 26 June 2017. The dry solid content of the activated sludge was 3.5 g/L. It was determined by weight measurements after drying at 105°C for 4 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):
28 d
Initial conc.:
20.1 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Mineral 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.

Experimental Set-Up
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.

Procedure
In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound and one reactor containing test item and reference compound (toxicity control) were set up.
12.9 mL activated sludge was filled up to 1500 mL with 1487.1 mL mineral medium corresponding to 30 mg/L dry solids. The system was sealed and aerated with CO2-free air overnight. The reactors were kept mixed with magnetic stirrers. On the next day, the absorber wash bottles were filled with 0.2 M NaOH and the test substance was added with inert PE-film as carrier material into the three test vessels and into the toxicity control vessel. The reference compound was added into the reference vessels and into the toxicity control vessel. The aeration rate was kept at a rate of 30 - 100 mL / min (1.6 - 5.5 bubbles / second) and determined visually daily on working days. The determination by counting the gas bubbles over a defined period using a stop watch was made on days 22 and 29. The CO2-free air production system, the air-tightness of the whole experimental set-up, the aeration of the absorber flasks and the magnetic stirrers were controlled daily on working days.
At the beginning of the study the IC concentration of the 0.2 M NaOH used for the CO2-absorption flasks was determined as 2.75 mg/L. The IC in the reactors at the beginning of the test was 0.74 mg/L. On the 3rd, 7th, 10th, 14th, 21st and 28th day 4 mL NaOH from the first of two CO2-absorber flasks connected in line was sampled and the IC's were determined. The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air. On the 28th day 2 mL of 4M hydrochloric acid (HCl) was added into each reactor to release the CO2 dissolved in water. On day 29 the IC was determined in both CO2-absorber flasks in line.

Evaluation of Data
The theoretical CO2 amount of the test item (ThCO2) is calculated as follows
ThCO2 [mg] = weight of added test item [mg] * carbon-content [mg/mg] * 44/12
The amount of CO2 released from the reactors is calculated through IC-measurements in the CO2-absorber flasks as follows:
CO2 [mg/1500 mL] = IC [mg/L]* Volumeabsorber flask [L] *44/12
Hereby the volume of the absorber flask at the beginning of the test was 0.2 litre (200 mL) and the volume was reduced through each sampling by 4 mL. The factor 44/12 corresponds the molar weight-ratio of CO2 and C.
The amount of CO2 removed for IC-measurement is considered by adding up the CO2 content of each sampling to the current CO2 content of each absorber flask:
CO2 total (i, x) = CO2 absorber flask (i, x) + CO2 sampling (i, x-1)
with
CO2 total (i, x) = Total amount of CO2 [mg] absorbed in the ith absorber flask at the xth sampling including the amount removed by sampling
CO2 absorber flask (i, x) = CO2 [mg] absorbed in the ith absorber flask derived from IC-measurement at the xth sampling.
 CO2 sampling (i, x-1) = Sum of CO2 [mg] removed from the ith absorber flask with the 1th to x-1thsampling (sampling volume 4 mL each CO2 removed = IC [mg/L] * 0.004 [L]*44/12]).
The percentage biodegradation is calculated from:
BiodegradationCO2 [%] = 100*(CO2 Test reactor [mg] - CO2 Blank reactor [mg])/ThCO2 [mg]
CO2 test reactor= Total CO2 evolution in the test reactor [mg]
CO2 Blank reactor = Total CO2 evolution in the blank reactor [mg] (mean of two vessels)
Reference substance:
benzoic acid, sodium salt
Remarks:
20 mg/L (organic carbon)
Preliminary study:
Not applicable
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
Test Item
No degradation could be observed within the test duration (28 days, after acidification). The biodegradation extent of the test item at the end of the test showed a negative value (-21.3%, mean of three replicates).

Toxicity control
The degradation extent in the toxicity control was 31.6% within 14 days: According to the criterion of the guideline (> 25 % degradation should be seen in toxicity controls) , the test substance had no/limited inhibitory effect on the inoculum.

Blank
The mean CO2 evolution of the blank flasks was 46.2 mg/L on day 28 after acidification.
Results with reference substance:
The reference compound sodium benzoate reached the pass levels for ready biodegradability within 7 days.

Ultimate biodegradation after x days (% of ThCO2)

Reactor

Day

0

3

7

10

14

21

28

29

11

Test Flasks

0

-3.9

-8.5

-9.7

-16.1

-23.0

-25.5

-26.6

12

0

-6.0

-12.5

-12.1

-18.2

-21.6

-21.9

-23.5

13

0

-2.9

-8.4

-8.9

-13.9

-15.1

-12.1

-13.7

4

Reference Flasks

0

52.4

70.4

82.4

81.2

79.7

79.5

74.0

5

0

52.2

65.0

79.8

76.8

77.1

74.8

69.7

6

0

48.7

61.9

72.1

76.8

75.7

80.3

75.6

14

Toxicity Control (test item + reference item)

0

27.9

31.4

34.9

31.6

29.9

31.2

28.9

Validity criteria fulfilled:
yes
Remarks:
See "Overall remarks".
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
No degradation of the test item could be observed within the test duration (28 days after acidification, mean of three replicates).
The test item did not meet the criteria for ready biodegradability (60% of ThCO2 within a 10-d window).
The degradation of the toxicity control after 14 days was 31.6%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.
Executive summary:

The ready biodegradability of the test item was investigated in a study conducted in accordance with OECD 301B (1992). No degradation of the test item could be observed within the test duration (28 days after acidification, mean of three replicates). The test item did not meet the criteria for ready biodegradability (60% of ThCO2 within a 10-d window).

The degradation of the toxicity control after 14 days was 31.6%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.

Description of key information

28d % biodegradation = 0% (not readily biodegradable); OECD 301B; Anon., 2018

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Anon., 2018 (OECD 301B)

The ready biodegradability of the test item was investigated in a study conducted in accordance with OECD 301B (1992). No degradation of the test item could be observed within the test duration (28 days after acidification, mean of three replicates). The test item did not meet the criteria for ready biodegradability (60% of ThCO2 within a 10-d window).

The degradation of the toxicity control after 14 days was 31.6%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.