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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:

24 October 2017 to 22 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge: The activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
- Date of collection: 25. Oct. 2017, batch no: 20171025.
- The sludge was filtrated, washed with medium 2x and re-suspended in test medium. It was then aerated until use.
- The dry matter was determined as 4560 mg suspended solids/L.

Duration of test (contact time):

28 d

Initial conc.:

33 mg/L

Based on:

test mat.

Initial conc.:

100.63 mg/L

Based on:

ThOD

Remarks:

with nitrification to nitrate

Initial conc.:

87.61 mg/L

Based on:

ThOD

Remarks:

without nitrification

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: The medium was freshly prepared (volumes were adapted to final volume needed in the test): Solution a 10 mL, Solution b 1 mL, Solution c 1 mL and Solution d 1 mL, H2O demin. ad 1000 mL.
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 and H2O demin. ad 1000 mL.
Solution c: Magnesium sulphate heptahydrate (MgSO4*7H2O) 22.5 g and 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 and H2O demin. ad 1000 mL.
- Solubilising agent: The test material was weighed directly into the test vessels.
- Test temperature: 20.5 – 20.7 °C
- pH: 7.2 – 7.4
- pH adjusted: No
- Aeration of dilution water: The sludge was filtrated, washed with medium 2x and re-suspended in test medium. It was then aerated until use.
- Suspended solids concentration: 25.0 mg/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 500 mL-flasks containing 250 mL test solution (toxicity flask 125 mL)
- 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).
- Number of culture flasks/concentration: 3
- Measuring equipment: OxiTop® measuring heads and controllers. Before the start of the test, the cartridge of the measuring heads was filled with soda lime to absorb CO2.

SAMPLING
- The oxygen which was consumed by the content of the vessels was measured automatically by the test apparatus.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 replicates (medium and inoculum)
- Abiotic sterile control: 1 replicate (test material and 10 mL 1 % HgCl2 solution/L)
- Toxicity control: 1 replicate (200 mg/L ThOD, 100 mg/L ThOD each of test material and positive control)
- Positive control: 3 replicates (60 mg/L sodium benzoate (corresponding to 99.86 mg/L ThOD), medium and inoculum)

THEORETICAL OXYGEN DEMAND
- With the formula of the compound given as CHClNNaOPS, and the molecular weight MR, the ThOD was calculated with the following equations:
- Equation for ThOD without nitrification:
ThOD NH3 = [16(2c + 0.5(h – cl – 3n) + 3s + (5/2)p + 0.5na – o)] / Mr
- Equation for ThOD with nitrification to nitrite:
ThOD NO2 = [16(2c + 0.5(h – cl) + 3s + (3/2)n + (5/2)p + 0.5na – o)] / Mr
- Equation for ThOD with nitrification to nitrate:
ThOD NO3 = [16(2c + 0.5(h – cl) + 3s + (5/2)n + (5/2)p + 0.5na – o)] / Mr

- ThOD Test Material: With the formula of the compound given as C24.1H49.94N3.33O1, and the molecular weight M = 402.44 g/mol, the following ThODs were calculated:
ThOD without nitrification: 2.67 mg/mg
ThOD with nitrification to nitrite: 3.07 mg/mg
ThOD with nitrification to nitrate: 3.20 mg/mg
The test material was directly weighed into the test vessels to get a concentration of 33 mg/L.

- ThOD Positive Control: With the formula of C7H5NaO2 and the molecular weight M = 144.1 g/mol, the following ThODs were calculated:
ThOD without nitrification 1.665 mg/mg
ThOD with nitrification to nitrite 1.665 mg/mg
ThOD with nitrification to nitrate 1.665 mg/mg
A stock solution of sodium benzoate containing 602.0 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 mg/L) was used.


CALCULATIONS
Oxygen Demand
The oxygen demand of test material and positive control vessels for each measuring point were calculated using the following equation:

O2 demand in mg/vessel = O2 demand in mg (vessel) – mean O2 demand in mg (blank controls)

For the abiotic control, the O2-demand of the blank controls was not subtracted.

Percentage Degradation
BOD = (mg O uptake by test material/positive control mg O uptake by blank) / (mg test material/positive control in vessel)
= mg O2 / (mg test material/positive control)

with
BOD = biological oxygen demand
mg O2 = O2-demand by oxidation of carbon

The percentage biodegradation is then obtained from:

% biodegradation = %ThOD = [BOD (mg O2 / mg test material/positive control) / ThOD (mg O / mg test material/positive control)] x 100 %

with
BOD = biological oxygen demand
ThOD = ThOD without nitrification

Biological degradation in % in the toxicity control is calculated following:

DT = [(OA – OB) / (ThODT *C + ThODP *CP)]*100 %

with:
OA = O2-uptake in the toxicity flask in mg O2/L at time t (oxidation of carbon)
OB = O2-uptake of the control in mg O2/L at time t (oxidation of carbon)
ThODT = Theoretical oxygen demand without nitrification test material in mg O2 / mg test material
ThODP = Theoretical oxygen demand without nitrification positive control in mg O2 / mg positive control
CT = Concentration of the test material in the toxicity flask in mg/L
CP = Concentration of the positive control in the toxicity flask in mg/L

Reference substance:

benzoic acid, sodium salt

Test performance:

All validity criteria were met with the exception of the degradation of the toxicity control:
- Degradation of positive control > 60 % (criteria: < 14 days) = 3 days, therefore valid.
- Mean O2 demand of the controls (criteria: < 60 mg/L) = 17.4 mg/L, therefore valid.
- Degradation in the toxicity flask on day 14 (criteria: >25 %) = 0 %, therefore toxic.
- pH at the end of the test (criteria: 6.0-8.5) = 7.1 - 7.7, therefore valid.
Oxygen consumption in the blank controls and in the positive controls was normal, indicating that the test system has worked properly. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Key result

Parameter:

% degradation (O2 consumption)

Value:

-6.6

Sampling time:

28 d

Remarks on result:

other: St. Dev not reported.

Details on results:

- Results are summarised in Table 1.
- The degree of biodegradation reached 0 % after 28 days. Therefore, when applying the 10-day-window, the test material is classified as not readily biodegradable following OECD 301F/EU C.4-D.
- If a test on the mixture is performed and it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test. As degradation missed 60 % in the course of the test the test material is considered as not readily biodegradable.
- The ThOD of the test material was calculated based on a theoretical formula of the test material, calculated as C24.1H49.94N13.33O1 based on elemental analysis. The sum of analysed elements (72.0 % C, 12.52 % H, 11.6 % N, 3.98 % O) was 100.1 %. The 10-day-window was not detected and during the test no degradation was observed.
- The test material can be stated as toxic towards the inoculum in a concentration of 34 mg/L. No degradation has taken place.
- The measured NO3 and NO2 concentrations were in a marginal range. Therefore, the oxygen consumed by nitrification can be stated as not significant.

Results with reference substance:

Degradation of positive control was required to be > 60 % <14 days, this criteria was met after 3 days. % biodegradation after 28 days was 86.7, 84.7 and 83.7 % for the three replicates.

Table 1: pH-values on Day 0 and Day 28 and Oxygen Demand and Degradation Values on Day 28

Flask	Blank Control			Positive Control			Test Material				Abiotic Control	Toxicity Control
	1	2	3	1	2	3	1	2	3	Mean
pH at day 0	7.4	7.4	7.4	7.4	7.4	7.4	7.4	7.4	7.4	-	7.2	7.4
pH at day 28	7.3	7.3	7.3	7.7	7.7	7.7	7.6	7.6	7.6	-	7.1	7.7
Oxygen demand in mg/L at day 28	19.8	18.0	14.4	104	102	101	9	14.4	9	-	0	14.5
Oxygen demand in mg/L after deduction	-	-	-	86.6	84.6	83.6	-8.4	-3.0	-8.4	-	0.0	-2.9
% biological degradation	-	-	-	86.7	84.7	83.7	-8.3	-3.0	-8.5	-6.6	0.0	-1.4

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

Under the conditions of this study the test material is not readily biodegradable.

Executive summary:

The biodegradation potential of the test material was investigated in accordance with the standardised guidelines OECD 301F and EU Method C.4-D, under GLP conditions. The test was performed under aerobic conditions in the Manometric Respirometry Test.

The test material was tested using a concentration of nominal 33 mg test material/L (equivalent to a theoretical oxygen demand of 100 mg O2/L). Activated sludge was used as inoculum. The test was left running for 28 days.

With the exception of the degradation of the toxicity control, all validity criteria were met. The positive control reached the pass level of 60 % on day 3 (criterion:≤14). If degradation in the toxicity flask is below 25 % after 14 days, the test material must be considered as toxic towards the inoculum. Because no degradation in the toxicity flask was observed at the end of the test, the test material can be stated as “toxic towards the inoculum in a concentration of 34 mg/L”.

The following data could be determined for the test material:

10-day-window: Not detected

Degradation at the end of 10-day-window; None

Degradation at the end of the test: 0 %

Pass level following guideline: 60 % at the end of 10-day-window for pure substances respective 60 % at the end of the test for mixtures.

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

If a test on the mixture is performed and it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test. As degradation missed 60 % in the course of the test material is considered as not readily biodegradable.

Under the conditions of this study the test material is not readily biodegradable.

Description of key information

Under the conditions of this study the test material is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

The biodegradation potential of the test material was investigated in accordance with the standardised guidelines OECD 301F and EU Method C.4-D, under GLP conditions. The test was performed under aerobic conditions in the Manometric Respirometry Test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested using a concentration of nominal 33 mg test material/L (equivalent to a theoretical oxygen demand of 100 mg O2/L). Activated sludge was used as inoculum. The test was left running for 28 days.

With the exception of the degradation of the toxicity control, all validity criteria were met. The positive control reached the pass level of 60 % on day 3 (criterion:≤14). If degradation in the toxicity flask is below 25 % after 14 days, the test material must be considered as toxic towards the inoculum. Because no degradation in the toxicity flask was observed at the end of the test, the test material can be stated as “toxic towards the inoculum in a concentration of 34 mg/L”.

The following data could be determined for the test material:

10-day-window: Not detected

Degradation at the end of 10-day-window; None

Degradation at the end of the test: 0 %

Pass level following guideline: 60 % at the end of 10-day-window for pure substances respective 60 % at the end of the test for mixtures.

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

If a test on the mixture is performed and it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results of the test. As degradation missed 60 % in the course of the test material is considered as not readily biodegradable.

Under the conditions of this study the test material is not readily biodegradable.