[No public or meaningful name is available]

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:

Experimental phase: 09 January 2018 to 07 February 2018. Report issued: 06 July 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Molecular formula:

not applicable as UVCB

IUPAC Name:

Fatty acids, C14-C18 and C18 unsaturated, amides with 2,2’-iminodiethanol

Test material form:

liquid

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 08 January 2018 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage. The activated sewage sludge sample was washed twice by settlement and re-suspension in mineral medium to remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection.

The mineral medium used in this study was that recommended in the OECD Guidelines. The deionised reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: Activated sludge
- Test temperature: 20 - 24 degrees centrigrade maintained in a temperature controlled room.
- pH: 7.4 - 7.5 at the start (day 0) and end of the test (day 28).
- pH adjusted: Yes, the pH was adjusted to pH 7.4 ±0.2 using diluted hydrochloric acid or sodium hydroxide solution
- Aeration of dilution water: Yes CO2 free air was continuous bubbled through the test solutions at a rate of 30 to 100 ml per minute per vessel and stirred continuously by magnetic stirrer.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 litre culture vessels
- Number of culture flasks/concentration: two replicates each for test item, inolculum control, procedure control, toxicity control.
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Details of trap for CO2 and volatile organics if used: Dreschel bottles containing 350 mL of 0.05 M NaOH to absorbe CO2 pproduced by degradation.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
- Measuring equipment: The samples were analysed for inorganic carbon using either a Shimadzu TOC-VCSH TOC analyzer or a Shimadzu TOC-LCSH TOC analyzer

CONTROL AND BLANK SYSTEM
- Inoculum control : Yes
- Procedure control: Yes
- Toxicity control: Yes

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

100

Sampling time:

28 d

Details on results:

The test item attained 100% 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 71% biodegradation after 14 days and 78% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 86% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 84% biodegradation was attained. The slight decrease in biodegradation between Days 14 and 28 was considered to be due to sampling/analytical variation. These results confirmed the suitability of the inoculum and test conditions and satisfied the validation criterion given in the OECD Test Guidelines.

Any other information on results incl. tables

The total CO2 evolution in the inoculum control vessels on Day 28 was 29.96 mg/L and satisfied the validation criterion given in the OECD Test Guidelines.

 

The inorganic carbon content of the test item suspension in the mineral medium at the start of the test was below 5% of the total carbon content and satisfied the validation criterion given in the OECD Test Guidelines.

 

The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and satisfied the validation criterion given in the OECD Test Guidelines.

 

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 a decrease in all replicate vessels with the exception of inoculum control Replicates 1 and 2 and procedure control Replicate 1. This decrease was considered to be due to sampling/analytical variation. 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.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test substance is considered to be readily biodegradable under the terms and conditions of OECD Guideline No. 301B.

Executive summary:

Introduction

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the following guidelines:

 

i)                   OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test"

ii)                 Method C.4-C of Commission Regulation (EC) No. 440/2008

iii)               US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

 

Methods

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 20 and 24 °C for 28 days.

 

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

 

Results

The test item attained 100% 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%.

 

Conclusion

The test substance is considered to be readily biodegradable under the terms and conditions of OECD Guideline No. 301B.