β,β-carotene

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 start date 10 October 2017 Experimental completion date 07 February 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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

β,β-carotene

EC Number:

230-636-6

EC Name:

β,β-carotene

Cas Number:

7235-40-7

Molecular formula:

C40H56

IUPAC Name:

β,β-carotene

Details on test material:

Identification:
Beta-Carotene
Batch:
WC01607211
Purity:
100.4%
Physical state/Appearance:
brown-red crystalline powder
Expiry Date:
19 January 2018
Storage Conditions:
approximately 4 °C in the dark, under nitrogen

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

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.

Preparation of Inoculum
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. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.5 g/L prior to use.
* Rinsed 3 times with 20 mL deionized reverse osmosis water prior to drying in an oven

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

5.6 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Experimental Design and Study Conduct

Preliminary Solubility Work
Information provided by the Sponsor indicated that the test item was insoluble in water. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test Item Preparation
In an initial experiment conducted at a concentration of 10 mg C/L, the toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25% biodegradation after 14 days. This result indicated that, under the strict terms and conditions of the OECD Guidelines, the test item would be classed as exhibiting inhibitory effects.
Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test item at a reduced concentration of 5 mg C/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. In error the test item was not weighed out or prepared under non actinic lighting. However the lighting in the laboratories where the test item was weighed out and prepared is LED lighting which does not emit UV light. Therefore it was considered to have had no adverse effect on the study.
The test item was dispersed directly in mineral medium.
An amount of test item (16.8 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 5.6 mg/L, equivalent to 5 mg carbon/L.

Reference Item Preparation
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium prior to the volume being adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Toxicity Control
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An amount of test item (16.8 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (approximately 7500 rpm, 15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 liters to give a final concentration of 5.6 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 15 mg carbon/L.

Preparation of Test System
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 5 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 15 mg carbon/L to act as a toxicity control (one vessel only).
Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 20 and 24 °C, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2300 mL of mineral medium and 36 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. If necessary the pH was adjusted to pH 7.4 ±0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2-free air.
The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer.
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.

Reference substance

Reference substance:

other: sodium benzoate

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

30

Sampling time:

28 d

Details on results:

Validation Criteria and Biodegradation
The total CO2 evolution in the inoculum control vessels on Day 28 was 29.96 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
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 and hence 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 hence 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 procedure control Replicate 2, test vessel Replicate 2 and the toxicity control. 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.
The test item attained 30% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The toxicity control attained 45% biodegradation after 14 days and 54% 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

Inorganic Carbon Values on Each AnalysisOccasion

 

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs1	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	1.52	1.75	1.40	1.75	1.75	1.40	1.63	7.93/1.75*	1.52	1.98	2.10	1.87	2.22	1.98
2	7.77	-	7.77	-	23.32	-	23.20	-	3.83	-	6.73	-	24.36	-
6	13.73	-	15.92	-	35.06	-	37.14	-	10.15	-	15.92	-	31.95	-
8	14.56	-	16.86	-	34.06	-	39.79	-	11.35	-	17.09	-	35.20	-
10	17.21	-	15.96	-	40.81	-	50.96	-	15.50	-	18.01	-	38.42	-
14	15.07	-	15.75	-	40.80	-	41.59	-	14.51	-	18.59	-	35.81	-
21	18.36	-	19.83	-	50.59	-	44.17	-	17.13	-	25.46	-	43.94	-
28	24.97	-	24.08	-	50.17	-	47.27	-	26.10	-	28.79	-	44.13	-
29	23.82	1.04	23.05	2.20	45.31	1.04	51.88	1.04	25.38	1.63	30.50	1.74	47.82	1.74

 

 R = Replicate

Abs = CO2absorber vessel

-  = No valuedetermined

* Results from analysis of frozen sample used in calculation of results as initial analysis result considered to be erroneous

 

Percentage BiodegradationValues

 

Day	Biodegradation (%)
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	52	0	37
6	71	0	38
8	71	0	43
10	98	1	49
14	86	8	45
21	94	15	55
28	81	19	44
29*	84	30	54

 

 

* Day 29 values corrected to include any carry-over of CO2detected in Absorber2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 30% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

Introduction

A 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 OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

Methods

In an initial experiment conducted at a concentration of 10 mg C/L, the toxicity control vessel, containing both the test item and sodium benzoate, attained less than 25% biodegradation after 14 days. This result indicated that, under the strict terms and conditions of the OECD Guidelines, the test item would be classed as exhibiting inhibitory effects.

Therefore, following the recommendations of the Test Guidelines, in the definitive test the test item at a reduced concentration of 5 mg C/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 30% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.