Phenol, paraalkylation products with C12-rich branched olefins derived from propene oligomerisation, reaction products with sulphur monochloride and decene, reaction products with Benzoic acid, 2-hydroxy-,C14-18 alkyl dervis., polybutenyl benzenesulphonic acid, carbon dioxide and calcium hydroxide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 2016 to Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, conducted in an equivalent manner to OECD 301B guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

yes

Remarks:

Inert Carrier used to enhance availability to inoculum and study extended to 60 days

Principles of method if other than guideline:

The study was conducted in general accordance to guideline OECD 301B. The test substance has low water solubility (<0.5 mg/L), therefore an inert carrier (silica gel) was used in order to increase the availability of the test substance to the inoculum. The study was extended to 60 days to ascertain whether the test substance was inherently biodegradable.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

Activated sludge was collected from the Easton Wastewater Treatment Facility, Easton, Maryland on January 08, 2016. The Easton facility treats predominantly residential wastes. The sludge was sieved using a 2-mm screen, adjusted to approximately 1,000 mg total suspended solids (TSS)/L with mineral media and then aerated at test temperature until use. A total suspended solids measurement and standard plate count were performed on the inoculum on the day of test chamber preparation. Plates were incubated at 20±3ºC for approximately 48 hours.

Duration of test (contact time):

ca. 60 d

Initial conc.:

10 other: mg Organic Carbon/L

Parameter followed for biodegradation estimation:

CO2 evolution

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

62.6

Sampling time:

60 d

Details on results:

The average biotic and abiotic cumulative percent biodegradation for EC 903-162-9 were 17.4% and 45.2% respectively.

Results with reference substance:

The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which an average of 94.1% of theoretical CO2 was evolved.

RESULTS AND DISCUSSION

Carbon Analysis

The measured total organic carbon (TOC) concentration of the reference substance stock solution was 396.9 mg C/L. The volume of stock solution used to dose the reference chambers was adjusted based on the measured TOC value so that approximately 10 mg C/L was delivered.

Observations and Measurements

The room temperature range recorded during the test was 18.4-22.1 ºC. The results of the standard plate count and TSS measurement performed on the inoculum were 2.0 x 104 colony forming units (CFU)/mL and 907 mg/L, respectively.

The control chambers evolved an average of 103.9 mg CO₂ over the test period. This value has been corrected for the amount of CO₂ in the trapping solution since potassium hydroxide solution, even when freshly prepared, contains carbonates. The amount of CO₂ evolved by the control chambers did not exceed the 40 mg/L (120 mg total) value considered the acceptable limit for CO₂ evolution tests (1,2).

The viability of the inoculum and validity of the test were supported by the results of the reference substance, sodium benzoate, from which an average of 94.1% of theoretical CO₂ was evolved. An average percent biodegradation of greater than 60% was achieved by Day 7, thereby fulfilling the criteria for a valid test by reaching the pass level by Day 14 (1). The average total cumulative percent biodegradation for EC 903-162-9 independent of base oil degradation was 62.6%. The average biotic and abiotic cumulative percent biodegradation for EC 903-162-9 were 17.4% and 45.2% respectively. EC 903-162-9 may not be considered readily biodegradable, since 60% TCO₂ was not achieved within 10 days of achieving 10% TCO₂

Validity criteria fulfilled:

yes

Interpretation of results:

other: The test substance partially degraded during the study, but did not meet the criteria for ready biodegradation.

Conclusions:

Evidence of ready biodegradability in a Carbon Dioxide Evolution Test is 60% TCO2 within the 28-day test period. In addition, the pass level must be reached within 10 days of achieving 10% TCO2. The average total cumulative percent biodegradation for EC 903-162-9 independent of base oil degradation was 62.6%. The average biotic and abiotic cumulative percent biodegradation for EC 903-162-9 were 17.4% and 45.2% respectively. EC 903-162-9 may not be considered readily biodegradable, since 60% TCO2 was not achieved within 10 days of achieving 10% TCO2.

Executive summary:

The ready biodegradability of EC 903-162-9 was determined by the Carbon Dioxide Evolution

Test Method (OECD Guideline 301B). Tests of ready biodegradability are stringent tests that provide

limited opportunity for acclimation and biodegradation to occur. In the CO₂ test, inoculated mineral

medium was dosed with a known amount of test substance as the nominal sole source of organic

carbon and aerated with CO₂-free air. The CO₂ produced from the mineralization of organic carbon

within the test chambers was displaced by the flow of CO₂-free air and trapped as K₂CO₃ in KOH

trapping solution. The amount of CO₂ produced by the test substance (corrected for that evolved by

the blank inoculum) is expressed as a percentage of the theoretical amount of CO₂ (TCO₂) that could

have been produced if complete biodegradation of the test substance occurred. The test contained a

blank control group, a reference group, a treatment group, a base oil group and an abiotic treatment

group. Each group contained two replicate test chambers. The blank control was used to measure the

background CO₂ production of the inoculum and was not dosed with a carbon source. The reference

chambers were dosed with sodium benzoate, a substance known to be biodegradable, at a concentration

of 10 mg C/L. The treatment and abiotic treatment groups were used to evaluate EC 903-162-9 at

10 mg C/L. The base oil group was used to evaluate the biodegradation of the base oil component of EC

903-162-9 and was dosed with the base oil at approximately the same concentration as the base oil

introduced to the treatment and abiotic treatment groups in the base oil/test material mixture. The

results indicated that the activated sludge inoculum was active, degrading the reference substance

94.1%. The average total cumulative percent biodegradation for EC 903-162-9 independent of base

oil degradation was 62.6%. The average biotic and abiotic cumulative percent biodegradation for EC

903-162-9 were 17.4% and 45.2% respectively.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with GLP and following OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A sample of activated sewage sludge was obtained from Haddington Municipal Sewage
Works (a local sewage processing plant situated in Haddington, East Lothian, UK, which
handles predominantly domestic sewage) on 02 July 2012. On arrival at the laboratory, the
sludge was allowed to settle before a portion of the overlying supernatant was siphoned off to
concentrate the solid sewage inoculant portion to the required level. The resultant sludge was
then shaken by hand to homogenise thoroughly. Sub-samples (5 mL) of the homogenised
sludge were removed and dried in an oven at approximately 105°C and the suspended solids
content determined as 2.24 g/L which is out with the required range of 3-5 g/L. The sample
was therefore allowed to settle before a portion of the overlying liquid was removed. The
remaining sample was homogenised and the solids content re-determined; a value of 3.28 g/L
was obtained.

After solids determination, the sludge was aerated until required for use. Prior to use, it was
allowed to settle for ca 40 min; portions of the supernatant were added to the bioreactors
(except abiotic controls) for use as the microbial inoculum.

Duration of test (contact time):

28 d

Initial conc.:

34.8 mg/L

Based on:

test mat.

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

25.5

Sampling time:

28 d

Results with reference substance:

The reference item was readily biodegradable under the conditions of the test as >80%
biodegradation was achieved by Day 10; this demonstrates the acceptable viability of the
inoculum.

EC-903-162-9, at a nominal addition rate of 20 mg TOC/L (34.80 mg test item/L), was not

readily biodegradable under the conditions of the test since a transition from 10 to 60%

biodegradation in a 10 day window during the 28 day test was not achieved.

The mean cumulative biodegradation of EC-903-162-9 at Day 28 was 25.5%. Comparison

with the abiotic control data indicated that a significant proportion (ca 73%) of the observed

biodegradation was the result of abiotic processes.

The TOC content of the toxicity control bioreactor was equivalent to the combined TOC of

the individual test and reference bioreactors. The cumulative biodegradation observed for the

toxicity control expressed in terms of the total TOC added was 60.6% which correlates well

with the mean (59.3%) cumulative biodegradation for the separate test (25.5% (mean)) and

reference (93.0%) item data. This close correlation indicates that EC-903-162-9 was not

inhibitory to the microbial inoculum under test conditions. This conclusion is supported by

the fact that the extent of biodegradation of the reference item is similar in the reference

bioreactor (93.0%) and toxicity control (95.5%; calculated by subtracting the mean

biodegradation of the test item (25.5%) from the biodegradation expressed in terms of

reference item addition only (121.0%).

The Day 28 and Day 29 data generated with the abiotic controls demonstrated that CO2 was

not released from the inorganic carbon content of EC-903-162-9 on acidification to ca pH 2.5.

Observations of the dispersion of the test item were difficult and inconsistent probably due to

the small quantity of test item added and the visual distortion of the bioreactor walls. It was

clear, however, that the test item was dislodged from the cover slips during the test; at Day 28

only the abiotic controls demonstrated the presence of some remaining test item, situated on

the inner walls of the bioreactors.

The test met the guideline validity criteria since:

• The mean CO2 evolution in the controls was <40 mg/L (mean = 12.6 mg/L) at Day 28.

• The differences of extremes of the replicate test bioreactors was within 20% (actual value

= 2.0%) at the end of the test.

• The reference item achieved 60% biodegradation by Day 14.

Validity criteria fulfilled:

yes

Interpretation of results:

other: Not readily biodegradable under test conditions

Conclusions:

The test item, EC-903-162-9, was not readily biodegradable under the conditions of the test.
A mean total of 25.5% degradation was achieved by Day 28.

Executive summary:

The ready biodegradability of EC-903-162-9 was assessed over a 28 day period using the CO2

Evolution (Modified Sturm) Test. The study was conducted in accordance with the

procedures outline in the OECD Guideline 301B.

The test was conducted at a nominal loading rate of 20 mg Total Organic Carbon (TOC)/L

(34.80 mg of EC-903-162-9/L, based on a determined total carbon content of 60.39% by weight).

The test item, EC-903-162-9, was not readily biodegradable under the conditions of the test.

A mean total of 25.5% degradation was achieved by Day 28. Therefore EC-903-162-9, was

not readily biodegradable under the conditions of the test as

it failed to achieve a transition from 10% to 60% degradation in a 10 day window during the

28 day test. The abiotic control data indicate that a significant proportion of the

biodegradation recorded for EC-903-162-9 was a result of abiotic degradation; the

acidification of the bioreactors on Day 28 did not result in the generation of CO2 from the

carbonate component of EC-903-162-9.

The test item, EC-903-162-9, was not considered to be inhibitory to the microbial inoculum

since the biodegradation observed in the toxicity bioreactor was similar to that expected from

the individual test and reference bioreactors.

Description of key information

In the key study (2016 OECD 301B), EC 903-162-9 was shown to undergo biotic and abiotic degradation, but did not fulfil the criteria for ready biodegradability. Due to the test items low water solubility, silica gel was used to enhance the bioavailability to the inoculum, the study was also extended to 60 days to investigate the inherent degradation potential of the test item. A mean total of 62.6% degradation was achieved after 60 days. The average biotic and abiotic cumulative percent biodegradation for EC 903-162-9 were 17.4% and 45.2% respectively. Based on these findings it was concluded that the test item was not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

In the key study (2016 OECD 301B), EC 903-162-9 was shown to undergo biotic and abiotic degradation, but did not fulfil the criteria for ready biodegradability. Due to the test items low water solubility, silica gel was used to enhance the bioavailability to the inoculum, the study was also extended to 60 days to investigate the inherent degradation potential of the test item. A mean total of 62.6% degradation was achieved after 60 days. The average biotic and abiotic cumulative percent biodegradation for EC 903-162-9 were 17.4% and 45.2% respectively. Based on these findings it was concluded that the test item was not readily biodegradable.

In the supporting study, the test item, EC-903-162-9, was not readily biodegradable under the conditions of the test. A mean total of 25.5% degradation was achieved by Day 28. Therefore EC-903-162-9, was not readily biodegradable under the conditions of the test as it failed to achieve a transition from 10% to 60% degradation in a 10 day window during the 28 day test. The abiotic control data indicate that a significant proportion of the biodegradation recorded for EC-903-162-9 was a result of abiotic degradation; the acidification of the bioreactors on Day 28 did not result in the generation of CO₂from the carbonate component of EC-903-162-9.

Although the supporting study was conducted to GLP standard and in accordance with OECD guideline 301B, the methodology used did not take account of the low water solubility of the test item, therefore the results are not considered indicative of the inherent biodegradation potential of the test item. As such the key study is considered to be more indicative of the inherent biodegradation potential of the test item.

It is concluded that the test item is not readily biodegradable, it does show degradation potential, the majority of which is abiotic given the high levels of inorganic carbon present in the substance. However, even using modifications to the standard protocol (extension to 60 days and silica gel) complete degradation was not achieved, illustrating that there are organic constituents within the UVCB substance that are not readily biodegradable.

No further degradation testing is proposed, this is in agreement with the specific rules of adaption text that is contained in column 2 section 9.2 Annex VIII of Regulation (EC) No 1907/2006, which states"Further biotic degradation testing shall be proposed by the registrant if the chemical safety assessment according to Annex I indicates the need to investigate further the degradation of the substance. The choice of the appropriate test(s) depends on the results of the chemical safety assessment."The CSA does not indicate the need for further degradation testing, therefore none is proposed.