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
Study period:
20 Feb-7 June 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:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
Appearance: Straw to yellow syrup
Purity/Composition: Not indicated
Test item storage: At room temperature

Additional information
Test Facility test item number: 209099/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Specific details on test material used for the study:
Batch P7728
DOM Nov 17 2016

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. After treatment the concentration of suspended solids (SS) was determined to be 3 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 3 mL per litre of mineral medium, leading to a SS concentration of 8 mg/L.
Initial test substance concentration
Initial conc.:
ca. 15.7 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
WICKENOL 535 was a straw to yellow syrup (UVCB) and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. A sample of the test item was taken for determination of the Total Organic Carbon (TOC) content. TOC analysis was performed using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu SSM-5000A (Solid Sample module for Total Organic Carbon Analyzer) (Shimadzu, Kyoto, Japan). Calibration standards were Glucose (C6H12O6, 99.5%, Sigma, Steinheim, Germany) as total carbon (TC) standard and Sodium carbonate (Na2CO3, p.a., Merck, Darmstadt, Germany) as inorganic carbon (IC) standard. The Total Organic Carbon (TOC) content of the test item was determined to be 76.23%. The test item was tested in duplicate at a target concentration of 15.7 mg/L, corresponding to 12 mg TOC/L. No correction was made for the purity/composition of the test item.
The test item was coated on silica particles. Aliquots of 37 µL were pipetted directly onto weighed amounts of silica in clear glass weighing bottles (for details, see Table 1). After careful mixing, the silica containing the test item was added directly to the 2 litre test bottles containing medium with microbial organisms and mineral components. Subsequently, Milli-RO water was added to each weighing bottle containing the silica in order to quantitatively add all silica to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.
Any residual volumes were discarded.
Set-Up of Test Vessels
Test vessel Volume of test item 1) Amount of Sodium acetate Amount of silica Volume of medium Concentration of test item ThCO2
Blank A - - - 2000 mL - -
Blank B - - - 2000 mL - -
Silica Blank A - - 1.00 g 2000 mL - -
Silica Blank B - - 1.00 g 2000 mL - -
Procedure Control - 79.9 mg - 2000 mL - 85.5 mg CO2/vessel
Bottle A 37 µL - 1.00 g 2000 mL 15.7 mg/L 88.1 mg CO2/vessel
Bottle B 37 µL - 1.00 g 2000 mL 15.7 mg/L 88.1 mg CO2/vessel
Toxicity Control 37 µL 79.9 mg 1.00 g 2000 mL 15.7 mg/L 173.6 mg CO2/vessel

1): Density: 0.85 g/mL

Test Procedure and Conditions
Test duration 28 days for the inoculum blank, silica blank, and test item (last CO2 measurement on day 29).
14 days for the procedure and toxicity control (last CO2 measurement on day 15).
During the test period, the test media were aerated and stirred continuously.
Test vessels 2 litre brown coloured glass bottles.
Silica gel High purity silica gel (SiO2) of a suitably small particle size was used at a maximum amount of 1 g per test vessel (Sigma Aldrich, Steinheim, Germany).
Milli- RO water Tap-water purified by reverse osmosis (Milli- RO) and subsequently passed over activated carbon.
Stock solutions of A) 8.50 g KH2PO4
mineral components 21.75 g K2HPO4
67.20 g Na2HPO4.12H2O
0.50 g NH4Cl
dissolved in Milli- RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli- RO water andmade up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli- RO water andmade up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli- RO water andmade up to 1 litre.
Mineral medium 1 litre mineral medium contains: 10 mL of solution (A),
1 mL of solutions (B) to (D) and Milli- RO water.
Barium hydroxide 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
Synthetic air (CO2 < 1 ppm) A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
Preparation of Bottles
Pre-incubation medium The day before the start of the test (day -1) mineral components, Milli- RO water (ca. 80% of final volume) and inoculum were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Type and number of bottles Test suspension: containing test item, silica, and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Silica blank: containing silica and inoculum (2 bottles)
Procedure control: containing reference item and inoculum (1 bottle).
Toxicity control: containing test item, silica, reference item, and inoculum (1 bottle).
Preparation At the start of the test (day 0), test item was added directly to weighed amounts of silica. Thereafter, silica with test item and reference item were added to the bottles containing the microbial organisms and mineral components. Silica without test item was added to the silica blanks.
The volumes of suspensions were made up to 2 litres with Milli- RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 mL
0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.
Determination of CO2

Experimental CO2 production The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
Measurements Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the blanks and test item. Titrations for the procedure and toxicity control were made over a period of at least 14 days.
Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers were moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 15 (procedure and toxicity control) and on day 29 (remaining vessels).
Theoretical CO2 production The theoretical CO2 production was calculated from the results of the TOC-analysis.
Measurements and Recordings

pH At the start of the test (day 0) and on the penultimate day (day 14 for the procedure and toxicity control and day 28 for the inoculum blanks, silica blanks, and test item), before addition of concentrated HCl.
Temperature of medium Continuously in a vessel with Milli- RO water in the same room.


Reference substance
Reference substance:
other: Sodium acetate

Results and discussion

Test performance:
The ThCO2 of WICKENOL 535 was calculated to be 2.80 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.
% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
>= 66
Sampling time:
28 d
Parameter:
% degradation (CO2 evolution)
Value:
53
Sampling time:
28 d

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
In conclusion, since the results of the present test indicate that the pass level criterion was almost fulfilled for bottle B the results can be used to indicate inherent biodegradability. However, based on the results of vessel A, WICKENOL 535 can be considered readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

The objective of the study was to evaluate the test item WICKENOL 535 for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with activated sludge.

The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992. At the request of the Sponsor, the test item was coated on silica particles. Additional blanks were included to correct for the possible effects of using silica.

WICKENOL 535 was a straw to yellow syrup (UVCB) and was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L. The Total Organic Carbon (TOC) content of the test item was determined to be 76.23%. Based on the TOC content the ThCO2of the test item was calculated to be 2.80 mg CO2/mg. The test item was tested in duplicate at a target concentration of 15.7 mg/L, corresponding to 12 mg TOC/L.

The study consisted of eight bottles:

·        2 inoculum blanks (no test item),

·        2 silica blanks (silica, no test item),

·        2 test bottles (silica, WICKENOL 535),

·        1 procedure control (sodium acetate) and

·        1 toxicity control (silica, WICKENOL 535, and sodium acetate).

Aliquots of 37 µL were pipetted directly onto weighed amounts of silica in clear glass weighing bottles. After careful mixing, the silica containing the test item was added directly to the 2 litre test bottles containing medium with microbial organisms and mineral components. Subsequently, Milli-RO water was added to each weighing bottle containing the silica in order to quantitatively add all silica to the test medium.The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.Test duration was28 days for the inoculum blank, silica blank, and test item (last CO2measurement on day 29) and 14 days for the procedure and toxicity control (last CO2measurement on day 15).

The relative biodegradation values calculated from the measurements performed during the test period revealed 66% and 53% biodegradation of WICKENOL 535, for A vessel and B, respectively (based on ThCO2). Furthermore, since the test item is a mixture of structurally similar chemicals and gradual biodegradation is anticipated, the 10-day window need not be applied.

In the silica blank, slightly higher background CO2production was observed. The test item bottles were compared with the silica blanks.

In the toxicity control, WICKENOL 535 was found not to inhibit microbial activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion,since the results of the present test indicate that the pass level criterion was almost fulfilled for bottle B the results can be used to indicate inherent biodegradability.[1]However, based on theresults of vessel A,WICKENOL 535 can be considered readily biodegradable under the conditions of the modified Sturm test presently performed.


 


[1]OECD Guidelines For The Testing Of Chemicals. Revised Introduction To The OECD Guidelines For Testing Of Chemicals, Section 3, Part 1, Chapter 2.5, Paragraph 36. (adopted July 23 March 2006).