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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-03 to 2018-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented guideline study with GLP and no deviations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
OECD Guideline 301 B / CO2 Evolution test (adopted July 17, 1992)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test item: Tall oil pitch, reaction product with triethylene glycol
Batch number: LABO 16-02
CAS name: Tall-oil pitch, ester with triethylene glycol
Purity: 100% (UVCB substance)
TOC*: 73.9%
Water solubility: Sparingly soluble
Appearance: Viscous (liquid), black
Expiry date: 2018-11-14
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Test system: Inoculum of the aqueous phase of non-adapted activated sludge
Source: Municipal sewage treatment plant, 31137 Hildesheim, Germany
Reasons for the selection: Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
Source: Municipal sewage treatment plant, 31137 Hildesheim, Germany
Receipt: 2017-08-16

Pre-treatment:
The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2.5 hours. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained at 19 – 22 °C in an aerobic condition by aeration with CO2 free air until test start (7 days). 10 mL/L of this mixture were used to initiate inoculation.

Colony forming units: Approx. 107 - 108 CFU/L in the test vessel
Duration of test (contact time):
28 d
Initial conc.:
14 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Duration: 28 days
Application: Once at test start
Test vessels: 5000 mL, brown glass
Volume of the test medium: 3000 mL
Test medium: Mineral salts medium acc. to OECD Guideline 301 B / CO2 Evolution Test
Test temperature: Nominal 22 ± 2 °C, Measured 20.0 – 24.0 °C
Dispersion treatment: Continuous stirring
Aeration: 30 - 100 mL/min (CO2-free air)
Photoperiod: Low light conditions (brown glass bottles)

The concentration of the test item and the theoretical CO2 production (ThCO2) were calculated based on the carbon content.

The following incubation vessels were prepared:
- two for the inoculum control (C1, C2)
- one for the functional control (R1)
- two for the test item concentration (P1, P2)
- one for the toxicity control (T1)

Additionally, one test item replicate (PIC) for IC measurement in the mineral medium was prepared without inoculation.

The necessary amounts of ultrapure water, mineral salts medium and inoculum were placed in each incubation vessel. The vessels were aerated for 24 h with CO2 free air. After 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.

840 mg test and 60 mg reference item were weighed out. The test item was dissolved in 10 mL of dichloromethane, 0.5 mL of the test item solution was pipetted onto 1.5 g sea sand on an object slide. For the inoculum control replicates with sea sand, the object slides with the sand were treated with 0.5 mL of dichloromethane. The sand was allowed to dry for at least one hour and then transferred together with the object slide into the test vessel. The reference item was transferred to the respective incubation vessel. The vessels were made up to 3 L with ultrapure water and connected to the system for the production of CO2 free air.

On day 28, 1 mL 37 % HCl was added to each of the vessels. Aeration was continued for further 24 h and the quantity of CO2 released was determined.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
14
Sampling time:
28 d
Details on results:
Carbon Content of the Test Item: Based on the carbon content a ThCO2 of 2.71 mg CO2/mg test item was calculated. A test concentration of 14 mg/L, corresponding to a carbon content of 10.4 mg C/L in the test vessels was selected.

Colony Forming Units of the Inoculum: Colony forming units (CFU) of the inoculum for the Modified Sturm Test were determined prior to test start by standard dilution plate count: approx. 1.31 x 10^9 CFU/L, corresponding to approx. 1.31 x 10^7 CFU/L in the test vessel.

The biodegradation of the test item in both replicates reached the 10% level (beginning of biodegradation) on day 18 and did not reach the 60% pass level within the 28-day-period of the study. The mean biodegradation on day 28 was 14%. Under the test conditions the test item is classified as not readily biodegradable within the 28 day period of the study.

The measured IC concentration was 86.92 µg C/L in the mineral medium with test item. Since the test item is insoluble, the nominal C-concentration was used (10.4 mg C/L). Accordingly, the IC content was 0.84%.
Results with reference substance:
The adaptation phase of the functional control changed within 4 days into the degradation phase (degradation >/= 10%). The course of the degradation was rapid and the functional control reached the pass level of 60% within 6 days and a maximum biodegradation of 88% on day 28. The validity criterion degradation >/= 60% after 14 days is fulfilled.

In the toxicity control containing both test and reference item a biodegradation of 46% was determined within 14 days, with 54% degradation after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

In the inoculum control the total CO2 production was 21.7 mg CO2/L after 28 days. In the inoculum control with sea sand the total CO2 production was 22.0 mg CO2/L after 28 days.

Table 2:   CO2-Production and Biodegradation after 28 Days

CO2-Production

Functional

Test Item

Toxicity Control

 

Control

 

test item +
reference item

1

2

Net        [mg/3 L]

113.1

17.3

15.0

129.5

[mg/L]

37.7

5.8

5.0

43.2

Theor.   [mg/3 L]

127.8

113.8

241.6

[mg/L]

42.6

37.9

80.5

Degradation [%]

88

15

13

54

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was reached 14% after 28 days and therefore is classified as not readily biodegradable.
Executive summary:

The biodegradability of Tall oil pitch, reaction product with triethylene glycol was determined by a modified Sturm Test and according to OECD giudeline 301B. The test item was reached 14% after 28 days and therefore is classified as not readily biodegradable.

Description of key information

The biodegradability of Tall oil pitch, reaction product with triethylene glycol was determined by a modified Sturm Test and according to OECD giudeline 301B. The test item was reached 14% after 28 days and therefore is classified as not readily biodegradable.

Key value for chemical safety assessment

Additional information