Sodium glyoxylate

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:

The study was conducted between 20 December 2016 and 22 February 2017

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium glyoxylate

EC Number:

220-298-8

EC Name:

Sodium glyoxylate

Cas Number:

2706-75-4

Molecular formula:

C2H2O3.Na

IUPAC Name:

sodium oxoacetate

Specific details on test material used for the study:

Identification: Safelink SPM-01
Physical state/Appearance: colorless to slightly yellow powder
Batch: G150201
Purity: 99.9%
Expiry Date: not supplied
Storage Conditions: room temperature in the dark

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
23 January 2017 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 2.6 g/L prior to use.
* Rinsed three 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.:

40 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Medium
The mineral medium used in this study was that recommended in the OECD Guidelines.

Experimental Design and Study Conduct
Preliminary Solubility Work
Information provided by the Sponsor indicated that the test item was soluble in water at a concentration of 17.1 wt% at 23 ºC. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Preliminary Work
In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge, the following work was conducted and samples analyzed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-VCPH TOC analyzer. During sample preparation, samples are either filtered or centrifuged to remove sewage sludge solids.
A nominal amount of test item (200 mg) was dissolved in mineral medium (1 liter) to give a 200 mg/L stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter). A further nominal amount of test item (200 mg) was dissolved in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 liter. Two samples were taken for DOC analysis; one after filtration through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 30 mg ss/L and then filtering or centrifuging as per the test item samples.

Test Item Preparation
An amount of test item (120.0 mg) was dissolved in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 liters to give a final concentration of 40.0 mg/L, equivalent to 10 mg carbon/L.

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 (120.0 mg) was dissolved in approximately 400 mL of mineral medium with the aid of ultrasonication (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 40.0 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 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 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 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 21 and 25 °C, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 34.6 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. 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/min 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

Preliminary study:

The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. However it appeared that the test item was not in complete solution in mineral media at the concentration of 200 mg/L as the DOC results were less than 80% of nominal. Therefore, for the purpose of the study, the test item was dispersed into the test system with the aid of ultrasonication and samples taken for DOC analysis were taken prior to the addition of the test item and filtered to remove the suspended solids present.

Test performance:

Validation Criteria
The total CO2 evolution in the inoculum control vessels on Day 28 was 37.22 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.

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

65

Sampling time:

28 d

Details on results:

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.

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 65% 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 test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

The toxicity control attained 69% biodegradation after 14 days and 70% 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 79% biodegradation after 14 days and 82% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

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
	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	2.10	3.85	1.98	1.75	4.20	3.50	1.75	1.75	1.75	3.50^*	1.17	1.40^*	1.75	1.75
2	8.70	-	8.93	-	27.38	-	26.10	-	18.56	-	17.05	-	34.10	-
6	15.92	-	15.92	-	41.52	-	38.17	-	34.83	-	34.14	-	51.67	-
8	18.58	-	19.03	-	40.94	-	40.94	-	37.27	-	39.56	-	57.68	-
10	19.72*	-	19.72^*	-	41.72^*	-	41.49^*	-	38.30^*	-	40.59^*	-	59.05^*	-
14	24.48	-	24.37	-	48.96	-	47.15	-	40.23	-	46.35	-	66.07	-
21	34.14	-	32.00	-	56.22	-	54.42	-	51.71	-	54.31	-	72.78	-
28	30.24	-	30.69	-	54.21	-	52.08	-	48.95	-	53.53	-	70.45	-
29	35.74	3.83	38.19	4.18	63.68	3.83	59.23	3.94	54.00	4.18	58.34	4.18	78.51	4.18

 

R₁– R₂          =   Replicates 1 and 2

Abs              =  CO₂absorber vessels

*                =  Results from re-analysis of original sample

Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	60	30	42
6	80	62	60
8	74	65	65
10	73	66	66
14	79	63	69
21	74	66	66
28	76	69	67
29*	82	65	70

*Day 29 values corrected to include any carry-over of CO₂detected in Absorber 2

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	10.03^**	-0.07	0
Test Item 10 mg C/L R2	10.22^**	-0.03	0

R₁– R₂     =  Replicates 1 and 2

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

**    Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 65% 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 test item can therefore 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; CO₂Evolution 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

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 21 and 25 °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 65% 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 test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.