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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:
09.07.2019-06.08.2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
Version 17.07.1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
30.05.2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
EssentÍal oil of Summer Savory/batch No. SSV1.ES.sample
Date of manufacture: 08.01.2019
Expration date of the batch: 08.01.2021
UVCB substance;
Purity test date: 18.01.2019
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Activated sludge from a biologic sewage treatment plant was used. The chosen plant is treating mostly household sewage.
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
Date of collection: 09. Jul. 2019, batch no: E20190709.
The sludge was filtrated, washed with medium 2x, and re-suspended in test medium. It was then aerated.
The dry matter was determined as 3.80 g suspended solids/L.
Duration of test (contact time):
28 d
Initial conc.:
100.48 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Sodium benzoate, CAS-No. 532-32-1 was used as positive control.
A stock solution of sodium benzoate containing 600.2 mg/L in demin. H2O was prepared. In the positive control flasks, a concentration of 60 mg/L sodium benzoate (corresponding to a ThOD of 100.10 mg/L) was used.
Activated sludge from a biologic sewage treatment plant was used. The chosen plant is treating mostly household sewage.
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
Date of collection: 09. Jul. 2019, batch no: E20190709.
The sludge was filtrated, washed with medium 2x, and re-suspended in test medium. It was then aerated.
The dry matter was determined as 3.80 g suspended solids/L.
All chemicals used in the test were “analytical grade” or >97.5%.

Stock Solutions
Solution a
Potassium dihydrogen phosphate (KH2PO4) 8.5 g
Dipotassium hydrogen phosphate (K2HPO4) 21.75 g
Disodium hydrogenphosphate dihydrate (Na2HPO4*2H2O) 33.4 g
Ammonium chloride (NH4Cl) 0.5 g
H2O demin. ad 1000 mL
The pH was 7.4.

Solution b
Calcium chloride (CaCl2) 27.5 g
H2O demin. ad 1000 mL

Solution c
Magnesium sulphate heptahydrate (MgSO4*7H2O) 22.5 g
H2O demin. ad 1000 mL

Solution d
Iron(III) chloride hexahydrate (FeCl3*6H2O) 0.25 g
Disodium ethylene-diaminetetraacetate dihydrate (Na2EDTA*2H2O) 0.4 g
H2O demin. ad 1000 mL

Test Medium
The medium was freshly prepared. 10 mL of solution a were mixed with 800 mL water, then 1 mL of solutions b, c and d were added and filled up to 1 L with H2O demin (volumes were adapted to final volume needed in the test).
Composition:
Solution a 10 mL
Solution b 1 mL
Solution c 1 mL
Solution d 1 mL H2O demin. ad 1000 mL

Mercury Chloride
HgCl2 (10 g/L), used for poisoning of abiotic flasks

Soda lime
CO2 adsorbent

Sodium Hydroxide, 1 M for pH-adjustment.

Test Vessels
500 mL-flasks were used as test vessels.
All glassware was cleaned with the laboratory cleaning agent and then rinsed with tap water (3x), diluted HCl (1x), tap water (3x) and deionised water (3x).

Reference substance:
benzoic acid, sodium salt
Test performance:
The medium was prepared using the stock solutions. The dry matter of the inoculum was determined. A sufficient number of test vessels was provided. A stock solution of the positive control containing 600.2 mg/L in deionised water was freshly prepared.
The test item was pipetted directly into the test vessels using the density (0.921 g/cm3).
Before the start of the test, the cartridge of the measuring heads was filled with soda lime and the program parameters (duration of the test, volume of the test flasks and volume of the test solution) were selected.

Experimental Parameters
Test item concentration: 35 mg test item/L (corresponding to 100.48 mg ThOD /L);
Positive control concentration: 60 mg /L sodium benzoate (corresponding to 100.10 mg/L ThOD);
Number of control flasks: 3 (medium and inoculum);
Number of positive control flasks: 3 (sodium-benzoate, medium and inoculum);
Number of test vessels: 3 (Summer savory oil, medium and inoculum);
Abiotic control: 1 (Summer savory oil and 10 mL 1% HgCl2 solution/L);
Toxicity control: 1 (200 mg/L ThOD, 100 mg/L ThOD each of test item and positive control nominal);
Volume of test vessels: 500 mL (600 mL total volume including measuring head);
Volume test solution: 250 mL (toxicity flask 125 mL);
Concentration of suspended solids 24.9 – 25.0 mg/L;
Temperature: 21.6 – 23.1 °C;
Duration: 28 days.

Start of the Test
The test vessels were filled with medium, then test item and positive control were added to the appropriate vessels and the pH was measured and adjusted to 7.2 – 7.3. After pH-measurement, the inoculum was added, the vessels were connected to the OxiTop® measuring heads and they were started via the OxiTop® Controller.

Performance of the Test
The oxygen which was consumed by the content of the vessels was measured automatically by the test apparatus.
After 28 days, the test was ended.
Because the test item and positive control do not contain nitrogen, presence of nitrate and nitrite was not checked.
Key result
Parameter:
% degradation (O2 consumption)
Value:
49
Sampling time:
28 d
Details on results:
The degree of biodegradation reached 49 % after 28 days.
The 10-day-window began on day 3 (this is stated as lag phase), at its end (day 13), 41 % were reached, staying below the pass level of 60 % given in the OECD guideline.
The degradation phase is defined as interval between the end of the lag phase and the reaching of 90 %. This was reached on day 18.
The degradation phase was from day 3 – day 18 (44 %).
Therefore, when applying the 10-day-window, the test item Summer savory oil is not readily biodegradable following OECD 301F/EU C.4-D.
Because the test item is a mixture the 10-day-window does not have to be taken into account. As degradation missed 60% in the course of the test, Summer savory oil is considered as not readily biodegradable, within 28 days, as well.

Oxygen Demand and Degradation Table

Flask

Blank

control 1

Blank

control 2

Blank

control 3

Positive control 1

Positive control 2

Positive control 3

pH at day 0

7.3

7.3

7.3

7.3

7.3

7.3

pH at day 28

7.0

6.9

6.9

7.1

7.1

7.1

Oxygen demand in mg/L at day 28

24.5

16.9

22.6

117

111

109

Oxygen demand in mg/L after deduction

--

--

--

95.7

89.7

87.7

% biological degradation

--

--

--

95.6

89.6

87.6

Table: pH-values on day 0 and day 28, oxygen demand and degradation values on day 28 (Blank and Positive Control)

Flask

Test item 1

Test item 2

Test item 3

Mean

Test item

Abiotic

Control

Toxicity Control

pH at day 0

7.3

7.3

7.3

--

7.2

7.3

pH at day 28

6.9

6.9

6.9

--

6.8

7.2

Oxygen demand in mg/L at day 28

71.5

88.4

69.6

--

0

141

Oxygen demand in mg/L after deduction

50.2

67.1

48.3

--

0.0

119.7

% biological degradation

49.9

*n.e.

48.0

49.0

0.0

60.2

Table: pH-values on day 0 and day 28, oxygen demand and degradation values on day 28(Test Item, Abiotic and Toxicity Control)

*n.e. = not evaluated

Due to a large deviation in the test replicate 2, and the other replicates were in the same range, the test replicate 2 was not used for evaluation

Validity

All validity parameters and values are presented in the following table:

Parameter

Criterion

Found

Assessment

Degradation of positive control≥60%

≤14 days

2 days

valid

Mean O2demand of the controls

< 60 mg/L

21.33 mg/L

valid

Degradation in the toxicity flask on day 14

> 25%

*58.0 %

valid

pH at the end of the test

6.0 – 8.5

6.8 – 7.2

valid

* Biological degradation of 58.0 % in the toxicity control on day 14 is calculated as follows:

DТ=[(OA -OB)/(ThODT*CT+ThODP*CP )]*100%

with

OA O2-uptake in the toxicity flask in mg O2/L on day 14 = 131 mg/L

OB Mean O2-uptake of the control in mg O2/L on day 14 = 15.7 mg/L

ThODT Theoretical oxygen demand without nitrification test item = 2.883 mg O2 / mg test item

ThODP Theoretical oxygen demand without nitrification positive control = 1.665 mg O2 / mg positive control

CT Concentration of the test item in the toxicity flask = 34 mg/L

CpConcentration of the positive control in the toxicity flask = 60 mg/l

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The following data was determined for the test item Summer savory oil:
10-day-window: day 3 – 13
Degradation phase day 3 - 18
degradation at the end of 10-day-window 41 %
degradation at the end of the test 49 %
Therefore, when applying the 10-day-window, Summer savory oil is classified as not readily biodegradable following OECD 301F/EU C.4-D.
Because the test item is a UVCB substance the 10-day-window does not have to be taken into account. As degradation missed 60% in the course of the test, Summer savory oil is considered as not readily biodegradable, within 28 days.
Executive summary:

All validity criteria were met.

Oxygen consumption in the blank controls and in the positive controls was normal, indicating that the test system has worked properly.

The ThOD of the test item was calculated based on a theoretical formula of the test item, calculated as C6.84H10.12O0.54. The formula was based on elemental analysis. The elemental analysis (C mean of 82.33 % and 82.09 % corresponds to 82.21 %, H 10.2 %, N < 0.2 %, O 8.61 %) was performed under non-GLP conditions by Mikroanalytisches Labor Pascher, An der Pulvermühle 1, 53424 Remagen, Germany. The sum of analysed elements was 101.02 %.

The 10-day-window began on day 3 (this is stated as lag phase), at its end (day 13), 41 % were reached, missing the pass level of 60 % given in the OECD guideline.

Due to a large deviation in the test replicate 2, and the other replicates were in the same range, the test replicate 2 was not used for evaluation. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Because the test item is a UVCB substance the 10-day-window does not have to be taken into account. As degradation missed 60% in the course of the test, Summer savory oil is considered as not readily biodegradable, within 28 days.

Description of key information

A Ready Biodegradability test has been performed according to OECD 301F / EU C.4-D Determination of the Ready Biodegradability of Summer savory oil under aerobic conditions in the Manometric Respirometry Test.

The test item Summer savory oil was tested using a concentration of 35 mg test item/L (equivalent to a theoretical oxygen demand of 100.48 mg O2/L). Activated sludge was used as inoculum. The test was left running for 28 days.

All validity criteria were met. The positive control reached the pass level of 60% on day 2 (criterion: 14).

The following data could be determined for the test item Summer savory oil:

 

10-day-window:                                                                               day 3 – 13

Degradation phase                                                                           day 3 - 18

degradation at the end of 10-day-window                                         41 %

degradation at the end of the test                                                       49 %

 

Therefore, when applying the 10-day-window,Summer savory oil is classified as not readily biodegradable following OECD 301F/EU C.4-D.

Because the test item is a UVCB substance the 10-day-window does not have to be taken into account. As degradation missed 60% in the course of the test,Summer savory oil is considered as not readily biodegradable, within 28 days.

Key value for chemical safety assessment

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

Additional information