N-2-hydroxyethyllactamide

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Aug - 21 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health -The Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

N-2-hydroxyethyllactamide

EC Number:

226-546-1

EC Name:

N-2-hydroxyethyllactamide

Cas Number:

5422-34-4

Molecular formula:

C5H11NO3

IUPAC Name:

N-2-hydroxyethyllactamide

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK (22 August 2016)
- Storage conditions: sludge was used on the day of collection
- Storage length: no storage
- Preparation of inoculum for exposure: activated sludge was washed twice by settlement and re-suspension in mineral medium. The washed sample was continuously aerated in the laboratory at a temperature of approximately 21 ºC.
- Pretreatment: The activated sludge was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC)
- Concentration of sludge: 30 mg suspended solids (ss)/L
- Determination of the suspended solids level: filtering 100 mL of the washed activated sewage sludge by suction through GF/A filter paper*. The filtertration was continued and filter was rinsed three times with 10 mL and dried in an oven at 105 ºC for at least 1 hour. The cold filter was weighed until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.
- Water filtered: yes, for the determination of suspended solid
- Type and size of filter used, if any: GF/A filter paper

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

23.2 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

10 mg/L

Based on:

other: carbon

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: mineral medium according to the guideline
- Test temperature: 22 to 24 °C
- Aeration of dilution water: 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 30 mL of inoculum and aerated overnight.
- Suspended solids concentration: equal to 3.0 g/L prior to use; test vessels were inoculated with the inoculum at a final concentration of 30 mg suspended solids (ss)/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: sealded 5 liter absorber vessels
- Number of culture flasks/concentration: 2 replicates
- Method used to create aerobic conditions: CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer.
- Measuring equipment: IC/TC ratio was measured using a Shimadzu TOC-VCPH TOC Analyzer
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules. The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.
- Other: On Day 28, 1 mL of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.

SAMPLING
- Sampling frequency: The first absorber vessels were sampled on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
- Sampling method: IC/TC ratio of the test substance (only Day 0): Samples (30 mL) were filtered through 0.45 μm Gelman AcroCap filters prior to DOC analysis. The samples were analyzed for IC and TC using a Shimadzu TOC-VCPH TOC Analyzer, 50 µL injected
IC Analysis: 2 mL of samples were taken from first CO2 absorber vessels. They were filtered through 0.45 μm Gelman AcroCap filters prior to DOC analysis.
- Sample storage before analysis: samples were analyzed for IC immediately; remainder of all samples were frozen for further analysis if required

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 Replicates (two absorber vessels per replicate)
- Procedure (Reference) control: 2 Replicates (two absorber vessels per replicate)
- Toxicity control: 1 Replicate (two absorber vessels per replicate)

STATISTICAL METHODS: no statistics

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

17.1 mg/L equivalent to 10 mg carbon/L

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (CO2 evolution)

Value:

99

Sampling time:

28 d

% Degradation 2

Parameter:

% degradation (CO2 evolution)

Value:

8

Sampling time:

6 d

% Degradation 3

Parameter:

% degradation (CO2 evolution)

Value:

23

Sampling time:

8 d

% Degradation 4

Parameter:

% degradation (CO2 evolution)

Value:

73

Sampling time:

10 d

Details on results:

Biodegradation:
Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item. The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of inoculum control Replicate 1, test item Replicate 1 and the toxicity control. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
The test item attained 99% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The toxicity control attained 89% biodegradation after 14 days and 77% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sludge.
Results in detail are summarized in tables 1 to 3 within section "Any other information on results incl tables"

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 69% biodegradation after 14 days and 88% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Validity criteria:

The total CO2 evolution in the inoculum control vessels on Day 28 was 30.72 mg/L. The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content. The difference between the values for CO2 production at the end of the test for the replicate vessels was <20%. All validity criteria given in the OECD Test Guidelines were fulfiled.

Table 1: Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75	1.75
2	6.61	-	6.85	-	21.34	-	20.65	-	7.77	-	7.66	-	16.12	-
6	12.80*	-	13.03*	-	27.91*	-	26.53*	-	16.26*	-	14.42*	-	17.30*	-
8	12.15	-	13.99	-	31.76	-	31.99	-	22.59	-	17.20	-	34.40	-
10	12.08	-	18.01	-	38.30	-	39.56	-	37.39	-	36.48	-	38.19	-
14	14.73	-	17.57	-	38.31	-	35.47	-	43.41	-	41.25	-	69.47	-
21	19.83*	-	19.83	-	42.36*	-	40.90	-	43.49	-	45.18	-	66.25	-
28	25.43	-	24.86	-	46.48	-	47.38	-	54.88	-	54.54	-	78.06	-
29	23.49	3.13	26.16	3.13	48.87	3.13	53.22	3.25	53.88	3.25	54.78	3.13	71.01	3.13

R1 – R2 = Replicates 1 and 2

Abs = CO2 absorber vessels

* = Re-analysis of remaining sample

Table 2: Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	48	3	16
6	48	8	7
8	63	23	36
10	80	73	39
14	69	87	89
21	73	82	77
28	73	99	88
29*	88	99	77

* Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Table 3: Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon* (mg/L)	Inorganic Carbon* (mg/L)	IC Content (% of TC)
Test Item 10 mg C/L R1	9.38	0.03	0
Test Item 10 mg C/L R2	10.00	-0.05	0

R1 – R2 = Replicates 1 and 2

* Corrected for control values. Negative values are due to measured concentrations being less than control values

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable