Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
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:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
temperature deviated on days 18 and 20 (24.8 C) above that stated in study plan (22+-2C). This deviation was considered to have not affected the integrity or validity of the study given that all validation criterion were met.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identification: OK (640-2) (Furoxy Hydroxy)
CAS Number: 56271-94-4
Physical state/Appearance: cream colored solid
Batch: G316533
Purity: not supplied
Expiry Date: 25 March 2018
Storage Conditions: approximately 4 ºC, in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Inoculum
A mixed population of activated sewage sludge micro-organisms was obtained on
4 December 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 2.8 g/L prior
to use.
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 10 mg/L
Based on:
TOC
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. The deionized 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).

Preliminary Solubility Work
Information provided by the Sponsor indicated that the test item was practically insoluble in
water. Therefore preliminary solubility/dispersibility work was performed in order to
determine the most suitable method of preparation. From the preliminary solubility work and following the recommendations of the
International Standards Organisation (ISO 10634, (1995)) and Handley et al (2002) it was
concluded that the best testable dispersions were found to be obtained when using the
ultrasonication, high shear mixing, and solvent with high shear mixing methods of
preparation. As there was no difference in the observations between these methods of
preparation it was considered appropriate to use ultrasonication in order to disperse the
test item into the test system.

Test Item Preparation
The test item was dispersed directly in mineral medium.
An amount of test item (63.6 mg) was dispersed 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 21.2 mg/L, equivalent
to 10 mg carbon/L.
A test concentration of 10 mg carbon/L was employed in the test following the
recommendations of the Test Guidelines.

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 and the volume 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 (63.6 mg) was dispersed in approximately 400 mL of mineral medium
with the aid of ultrasonication (approximately 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 21.2 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 20 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 32.1 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:
benzoic acid, sodium salt

Results and discussion

Test performance:
The total CO2 evolution in the inoculum control vessels on Day 28 was 30.38 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
(see Table 3) 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 an increase in all replicate vessels with the exception of inoculum control
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.

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
ca. 13
Sampling time:
28 d
Details on results:
The test item attained 15% 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 56% 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 111% biodegradation after 14 days with greater than 60%
degradation being attained in a 10-Day window. After 28 days 115% biodegradation was
attained. Biodegradation values in excess of 100% were 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.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 15% biodegradation after 28 days and therefore cannot be considered
to be readily biodegradable under the strict terms and conditions of OECD Guideline
No. 301B.