DL-alanine

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:

2018-02-15 - 2018-03-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

yes

Remarks:

EDTA free medium, The abiotic unit was disconnected after infection was seen in the incubation vessel,The test stopped after 3 weeks

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Remarks:

collected at a domestic wastewater treatment plant

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): The inoculum was freshly collected from the domestic sewage water treatment plant (SWTP) of the city of Geel on 9/02/2018 (Sludge 18 001)
- Method of cultivation: In the lab the sludge was allowed to settle and the supernatant was removed and replenished with 1 L of tap water. After resuspension, the sludge was centrifuged for 10 min (3000 rpm). The supernatant was removed and again replenished by 1L tap water. This step was repeated once more with mineral medium. After the last washing step, the active sludge suspension was placed in diffuse light at room temperature and aerated until use.
- Storage length: 5 days
- Preparation of inoculum for exposure: On 14/02/2018 a part of the sludge was centrifuged (10 min; 3000 rpm), the supernatant was removed and was replenished by BOD medium. The suspended solids (SS) content was estimated by the weight of material that is removed by filtration (0.22 pm) from a known volume of the homogenized sludge suspension.
Suspended solids content was 73 g/L.
- Concentration of sludge: 41 ml of this suspension was diluted with 959 mLof the mineral medium to provide a sludge suspension with 3 g/L dry weight. This diluted suspension was aerated until use.
- Initial biomass concentration: 3 g/L dry weight
- Water filtered: yes
- Type and size of filter used, if any: 0.22 µm

Duration of test (contact time):

20 d

Initial conc.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: BOD medium was prepared freshly on 14/02/2018 according to SOP TREAG015 v3, except in stock solution D no EDTA was added (replaced by 50 µL HCI (30%)). See Table 1 for composition
- Test temperature: room temperature (min-max: 19.5 - 22.0°C)
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: 73 g/L.
- Continuous darkness: diffuse light


TEST SYSTEM
- Culturing apparatus: 5 L brown glass incubation container and 2 small glass bottles filled with 200 mL 0.05N NaOH (NaOH traps 1 and 2)
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: a stopper with an air inlet
- Test performed in closed vessels due to significant volatility of test substance: 5 L brown glass incubation container
- Details of trap for CO2 and volatile organics if used: 2 small glass bottles filled with 200 mL 0.05N NaOH (NaOH traps 1 and 2). Incubation container and first NaOH trap are provided by a stopper with an air inlet and outlet and connected in series by tubing from incubation container to NaOH trap 1 and subsequently to NaOH trap 2. CO2 free air is entering the unit flowing first through the incubation compartment and next through NaOH-traps 1 and 2. In this way, the CO2 that is produced by biodegradation of the carbon sources in the incubation container is transported to the NaOH compartment, where it is trapped as inorganic carbon (NaHCO3)
Other: On 14/02/2018 the incubation containers were filled:
• Vessels 1 to 6: 2.4 L medium and 30 mL inoculum (90 mg/ SS)
• Vessel 7: 2.4 L medium
The containers and the remaining medium were aerated with CO2 free air overnight, to remove CO2 from the medium.
In vessels 3 to 7, the organic test item and reference item are the sole sources of organic carbon. In the control vessels (1 and 2) no organic nutrient source is present (background control).
After filling the incubation containers 14 bottles were filled with 200 ml of 0.05N NaOH and connected to the incubation containers and aeration was started.

SAMPLING
- Sampling frequency: every 2 to 3 days
- Sampling method: from NaOH traps
- Sterility check if applicable: yes

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes

RESULTS AND CALCULATION:
Chemical data are reported as mg/L C in the NaOH solution.
Using the volume in the NaOH trap the total amount of carbon that has been produced in the incubation vessel and was transferred to the NaOH trap can be calculated.
The mean amount of carbon that was found in the control units is used as the background value and is subtracted from the amount of carbon that is found in the other units.
By comparing the net amount of carbon in the NaOH trap to the amount of C that was initially loaded to the incubation vessel, the percentage of the theoretical CO2 production (ThCO2) can be calculated.
An excel sheet template was used for the calculations.
The 10-days window starts when the degradation level has reached 10% of ThCO2. The pass levels is 60% of the ThCO2 within 10 days after the start of the 10-days window.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Not performed.

Test performance:

Test conditions: diffuse light, room temperature (min-max: 19.5 - 22.0°C).
Sampling: 2 mL samples were taken from the NaOH trap 1 vessels using a disposable syringe, and immediately collected in 2 mL brown glass vials without headspace. Samples were stored refrigerated until analyses.
At the start of the test, a sample was taken from the NaOH 0.05 N stock solution (t=0) as a starting point.
Samples were taken from the NaOH traps on 16/02, 19/2, 21/2, 23/2, 26/2, 28/2, 2/3, 5/3 and 7/3.
Chemical analyses were performed each week on the collected samples.
On 2/3/2018 the abiotic unit was disconnected as from 28/02 on clearly a mold infection was visible in the incubation container, despite the HCI inhibitor.
As the results showed a plateau phase, it was decided to stop the test after three weeks.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 75

Sampling time:

16 d

Parameter:

% degradation (CO2 evolution)

Value:

60

Sampling time:

11 d

Parameter:

% degradation (CO2 evolution)

Value:

>= 10

Sampling time:

4 d

Details on results:

The test item is readily biodegradable. The 10-days window started on day 4 (>10% of ThCO2) and the pass level of 60% was reached on day 11. A plateau was reached around 75% of ThCO2 on day 16.

Results with reference substance:

The reference item reached the 10-days window on day 4 (>10% of ThCO2) and the pass level of 60% was reached on day 13. A plateau was reached around 65% of ThCO2 on day 13.
Details on rawdata and calculation attached in background material.

Validity criteria

• The total CO2 evolution in the inoculum blank at the end of the test was 19.3 mg/L CO2 medium, which is well below the maximum allowed value of 40 mg/L medium.

• The mean difference of extremes of replicate values of the removal of the test chemical at the plateau was 12.7% which is well below the maximum allowed value of 20%.

• The percentage degradation of the reference item reached the pass levels (CO2 production > 60% of ThCO2) on day 13, which is within 14 days as required by the OECD guideline.

• The biodegradation level in the toxicity control was 48% on day 13: the test item is therefore not considered as being inhibitory to the inoculum.

All validity criteria were met.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item is readily biodegradable. The 10-days window started on day 4 (>10% of ThCO2) and the pass level of 60% was reached on day 11. A plateau was reached around 75% of ThCO2 on day 16.

Executive summary:

This test aimed to evaluate the ready biodegradability of the test item DL-Alanine in water in aerobic conditions according to OECD Guideline 301B (1992) - CO₂-evolution test and in compliance with GLP criteria.

The amount of test and reference item added to the test vessels represented 20 mg/L C.

The inoculum (active sludge) was collected at a domestic wastewater treatment plant (City of Geel, Aquafin station). The sludge was washed and kept in the lab in low carbon conditions during 6 days. In the test vessels, 30 mg/l DW was used. The temperature was set to 19.5 - 22.0°C at diffuse light conditions.

The CO₂ produced in the test vessels was trapped In a 0.05N NaOH solution. Samples were taken from the NaOH traps every 2-3 days for inorganic carbon (IC) measurements. The duration of the test was 20 days (started on 15/02/2018 - ended on 7/03/2018). All validity criteria were met.

The 10-days window started on day 4. The pass level (60% of ThCO₂) was reached on day 11 and the plateau value (around 75% of ThCO₂) was reached on day 16.

It has been concluded that DL-Alanine is ready biodegradable.

Description of key information

DL-alanine was observed to be readily biodegradable by Weltens (2018). As could be expected, was L-alanine also found to be readily biodegradable.

Biowin correctly predicts the ready biodegradability of DL and L-Alanine (BIOWIN Version 4.10) Both have the same SMILES code.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater

Additional information

The ready biodegradability of DL-Alanine in water under aerobic conditions was determined according to OECD Guideline 301B (1992) - CO₂-evolution test and in compliance with GLP criteria.

The amount of test and reference item added to the test vessels represented 20 mg/L C.

The inoculum (active sludge) was collected at a domestic wastewater treatment plant (City of Geel, Aquafin station). The sludge was washed and kept in the lab in low carbon conditions during 6 days. In the test vessels, 30 mg/L DW was used. The temperature was set to 19.5 - 22.0°C at diffuse light conditions.

The CO₂ produced in the test vessels was trapped in a 0.05 N NaOH solution. Samples were taken from the NaOH traps every 2-3 days for inorganic carbon (IC) measurements. The duration of the test was 20 days (started on 15/02/2018 - ended on 7/03/2018). All validity criteria were met.

The 10-days window started on day 4. The pass level (60% of ThCO₂) was reached on day 11 and the plateau value (around 75% of ThCO₂) was reached on day 16.

Based on these results DL-Alanine is considered ready biodegradable.

Three other supporting studies were considered to be less reliable, because their documentation was limited. The experimental results of these three supporting studies also consistently indicate that L-alanine is readily biodegradable.