Carnosine

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:

22 May - 20 Jun 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)

Remarks:

Swiss Federal Office of Public Health, Bern, Switzerland

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: aeration stage of the local wastewater treatment plant, ARA Birs, Birsfelden, Switzerland
- Storage conditions: During the holding period of 1 d prior to use, the sludge was aerated at room temperature.
- Storage length: 1 d
- Preparation of inoculum for exposure: The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in tap water and finally in mineral medium. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined.
- Concentration of sludge: Based on the measured dry weight of the suspended solids, a calculated amout of wet sludge was suspended in mineral medium to obtain a concentration equivalent to 4 g dry material per liter, which was stored during 1 d. Prior to use, the dry weight of the sludge was again determined and the sludge was diluted with mineral medium. Defined volumes of this diluted activated sludge were then added to the mineral medium in the test vessels to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

113 other: mg O2/mg test item

Based on:

ThOD

Remarks:

ThODNH4

Initial conc.:

>= 226 - <= 227 other: mg O2/mg test item

Based on:

ThOD

Remarks:

ThODNO3

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral medium according to guideline
- Test temperature: 22 - 23 °C
- pH: 7.4 - 7.5 (test start), 6.5 - 7.8 (test end)
- pH adjusted: The pH of the mineral medium was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution.
- Suspended solids concentration: 30 mg dry material/L (final concentration)
- Continuous darkness: The test vessels were incubated under diffused lighting.
- Other: The test was conducted in a thermostatic cabinet.
- Determination of Nitrification: Since the test item contains nitrogen, nitrification with formation of nitrite and/or nitrate may occur. Therefore, at the end of the test, the nitrite and nitrate concentrations were measured in the test media containing the test item and in the inoculum controls. Prior to measurement, the samples were filtered through a nylon filter (pore size 0.45 μm).

TEST SYSTEM
- Culturing apparatus: Glass bottles with a nominal volume of 500 mL, test vessels contained 164 mL of test suspension, a suitable volume to assure sufficient oxygen in the headspace.
- Number of culture flasks/concentration: 2
- Measuring equipment: OxiTop Control system from WTW GmbH, Weilheim, Germany consisting of pressure measuring heads, test bottles, an inductive stirring system and the OxiTop OC 110 Controller.
- Details of trap for CO2 and volatile organics if used: Carbon dioxide was absorbed by sodium hydroxide pellets.
The prepared test vessels were closed air-tight with the OxiTop measuring heads.
- Other: The test temperature was recorded throughout the study at least on each working day. The pH was measured prior to the start of the test (Day 0) in each test vessel before the addition of the inoculum. At the end of incubation, the pH was measured again in every test vessel.
- Prepration of test vessel: 16.4 mg test item were added to the designated test vessels with mineral medium. No emulsifiers or solvents were used. Finally, activated sludge was added to each test vessel. The final test volume was 164 mL per test vessel.

SAMPLING
- Sampling frequency: The pressure reduction was continuously detected by the OxiTop pressure measuring heads and recorded every 3 h.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Mineral medium only (2 replicates)
- Toxicity control: 100 mg/L test item (ThODNH4: 113 mg/L) + 100 mg/L reference item (ThOD: 167 mg O2/mg) in mineral medium (1 replicate).
- Procedure control: 100 mg/L reference item (ThOD: 167 mg O2/mg) in mineral medium (2 replicates).

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (O2 consumption)

Value:

91

Sampling time:

28 d

Remarks on result:

other: based on ThODNO3

Parameter:

% degradation (O2 consumption)

Value:

85

Sampling time:

28 d

Remarks on result:

other: based on ThODNH4

Results with reference substance:

In the procedure controls, the reference item sodium benzoate was degraded by an average of 67% and 87% by Day 3 and 14, respectively, thus confirming the suitability of the activated sludge (≥ 60% degradation by Day 14). By the end of the test (Day 28), average biodegradation was 91% based on ThOD.

Results:

Nitrification:

The concentrations of nitrite-N were below the limits of detection (0.01 mg NO2 --N/L) in all test vessels.The nitrate concentrations in the two replicates containing the test item, and in the toxicity controls were 104, 106 and 107 mg NO3 -/L, respectively (after correction for the inoculum). This corresponds to 108, 109 and 111 mg O2/L, consumed in the formation of nitrate (using the conversion factor from NO3 - to O2 of 1.033). The analysis were performed at the end of the test, on Day 28 only. It is assumed that on Day 0 of the test the nitrite and nitrate amounts are negligible. In conclusion, significant nitrification had occurred in the test media containing the test item during the test period.The measured BOD in the test vessels containing the test item was corrected, according to the guidelines, for the oxygen consumed in the nitrification process and the resulting value was compared to the ThODNH4 in order to calculate the corrected biodegradation values for Day 28.

Test Item

The oxygen consumption of the test item in the test media rapidly increased from Exposure Day 1 until Day 8, when a mean biodegradation value of 80% of the ThODNO3 (or 160% of the ThODNH4) was reached. At the end of the test, on Day 28, the mean biodegradation value was 91% of the ThODNO3. Based on the ThODNH4 the mean biodegradation value at the end of the test was 181% and after correction for the amount of oxygen used in the nitrification process resulted in a mean biodegradation value of 85%. The pass level for ready biodegradability, i.e. biodegradation of at least 60% of the ThOD in 10-day window within the 28-day period of the test, was reached. In conclusion, the test item was found to be readily biodegradable under the test conditions within 28 days.

 

Toxicity Control

In the toxicity control, the run of the curve of the oxygen consumption over the 28-day exposure period correlated with the oxygen demand of the two added substances, i.e. test item and reference item. Within 14 days of exposure, biodegradation amounted to 89% and 124 % based on the ThODNO3 and the ThODNH4, respectively, reaching 93% and 131% at the end of the test. After correction of the BOD on Day 28 for the amount of oxygen used in the nitrification process, the biodegradation value based on the ThODNH4 resulted in 91%. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge micro-organisms at the tested concentration of 100 mg/L because biodegradation in the toxicity control was > 25% within 14 days of incubation.

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item was found to be readily biodegradable after 28 days of exposure to activated sludge under the conditions of the conducted Manometric Respirometry Test OECD 301F. Biodegradation reached a mean value of 91% based on the ThODNO3 and 85% based on ThODNH4 (after correction for the oxygen consumed by the nitrification process; without correction 181%) after 28 d.

Description of key information

Readily biodegradable: 91% (based on ThODNO3) and 85% (based on ThODNH4) after 28 d (O2 consumption, OECD 301F).

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

One GLP guideline study according to OECD 301 F is available investigating the ready biodegradability of the substance. Non-adapted, domestic activated sludge from a sewage treatment plant was used as inoculum with a nominal concentration of 100 mg/L test substance (corresponding to 113 mg/L based on ThOD NH4) and incubated for 28 d under diffused lighting. An inoculum blank, a procedure control (with 10.0 mg reference substance/L) and a toxicity control were run in parallel. The degradation rate of the test substance was 91% (based on ThODNO3) and 85% (based on ThODNH4). The reference substance was degraded to 91% after 28 d confirming the suitability of the inoculum. The test item is considered to be non-inhibitory, since the toxicity control attained 89 % degradation by Day 14 (criterion:≥25% degradation by Day 14).Since the test substance reached the pass level for ready biodegradability of > 60% removal of ThOD within 28 d it is readily biodegradable according to the OECD criteria.