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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from a study conducted according to OECD guideline, however no detailed description of study performance is given.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
not specified
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
28 d
Initial conc.:
393 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Study was conducted according to OECD guideline 301F
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Parameter:
BOD5
Value:
0 mg O2/g test mat.
Validity criteria fulfilled:
not specified
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
In a study conducted according to OECD guideline 301F the test substance Silastol H 200 was proven to be not readily biodegradable; 0% degradation was observed, determined by oxygen consumption.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-11 - 2016-05-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Specification
Activated sludge from a biologic sewage treatment plant was used. The chosen plant is treating mostly domestic sewage.
Source and Pre-Treatment
Source
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf.
Date of collection: 19. Feb. 2016, batch no: 20160219.
Pre-Treatment
The sludge was filtrated, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use. The dry matter was determined with 4060 mg suspended solids/L.
Duration of test (contact time):
77 d
Initial conc.:
17.3 mg/L
Based on:
test mat.
Remarks:
First test series
Initial conc.:
17.3 mg/L
Based on:
test mat.
Remarks:
Second test series
Initial conc.:
36.1 mg/L
Based on:
test mat.
Remarks:
Third test series
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The study was performed to examine the potential for aerobic biodegradation of the test item. For this aim 3 test series were performed.
First test series:
The test item (pure) Silastol H 200-TS was tested using a concentration of nominal 10 mg organic carbon/L (corresponding to 17.3 mg Silastol H 200-TS/L) and nominal 20 mg or-ganic carbon/L (corresponding to 34.5 mg Silastol H 200-TS/L) in test medium following OECD 301B and EU-Method C.4-C respectively.
To achieve a maximum bioavailability the test substance was dispersed as a thin layer on the surface of the test vessels, the nominal load of the test item Silastol H 200-TS was added into glass beakers and dissolved in a minimum amount of methanol. This solution was transferred into 2000 mL-SCHOTT-flasks. The glass beaker was rinsed twice with 5 mL methanol to guarantee for complete transfer of test item into the test vessel. Then, metha-nol was evaporated completely using compressed air. Thus, a thin layer of test item on the surface of the test vessels was achieved.
On the day of the start of the test, CO2-free medium and inoculum was filled into the test flask, see chapter 7.1.
In the blank control replicates the same amount of solvent without test item was added and completely evaporated.
Second test series:
The test item (pure) Silastol H 200-TS was tested using a concentration of nominal 10 mg organic carbon/L (corresponding to 17.3 mg Silastol H 200-TS/L) and nominal 20 mg or-ganic carbon/L (corresponding to 34.5 mg Silastol H 200-TS/L following OECD 301B and EU-Method C.4-C respectively.
In the second test series the test item Silastol H 200-TS was homogenised in a glass beaker filled with an aliquot of CO2-free test medium (10 mL) using an ultrasonic bath until a homogeneous dispersion was reached and transferred into the test vessels. The beaker was rinsed three times with 10 mL CO2-free test medium.
Third test series:
The test item dispersion Silastol H 200 was tested using a concentration of nominal 10 mg organic carbon/L (corresponding to 36.1 mg dispersion Silastol H 200/L) and nominal 20 mg organic carbon/L (corresponding to 72.1 mg dispersion Silastol H 200/L) in test me-dium following OECD 301B and EU-Method C.4-C respectively.
In this third test series the test item dispersion Silastol H 200 was weighed directly into the test flasks according to the amount of organic carbon.
Aniline was chosen as positive control.
Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L). To examine the potential for aerobic biodegradation of the test item, the test was left run-ning for 77 days.
Preparations
The medium was prepared from the stock solutions. The stock solution of the positive con-trol was prepared and its TOC was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined.
The test vessels were filled with medium and inoculum and the flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.
On the day of the start of the test, CO2-free medium and inoculum were filled into the test flask of the first test series. For the second and the third test series the test item was add-ed into the test flasks.
Flask volume 1500 mL
Apparatus blanks: 2, containing mineral medium only
Blank Controls: 2, containing mineral medium and inoculum
Positive control flasks: 2, containing positive control, mineral medium and inoculum
Test flasks: 2, containing test item, mineral medium and inoculum (for each concentration 10 and 20 mg/L TOC)
Inoculum concentration 25.0 mg/L
Temperature 19.1 – 21.6 °C
Duration 77 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L and for the test flasks additionally 10 organic carbon/L.
The apparatus blanks are used to determine traces of CO2 in the air supply. In this study no toxicity control and no abiotic control is required, because in another biodegradation test according to OECD 301B with Silastol H 200-TS (LAUS study 11121602G605) degra-dation in the toxicity flask was 39 % after 14 days. Therefore, the test item can be stated as “not toxic” towards the inoculum. Further, no abiotic degradation was observed in that study.
Apparatus
The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, mois-tened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.
Magnetic stirrers were used to prevent deposition of inoculum.
The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask.
Sampling
From each front scrubber flask, 13 samples were taken in order to determine the emitted CO2 (on day 0, 2, 4, 7, 9, 11, 14, 18, 23, 29, 37, 44 and 78). From the front scrubber flasks of the treatments listed below additional samples were taken on day 51, 58 and 65. The sample volume was 1 mL. The resulting change in the volume of the front flask was con-sidered in the calculation of emitted CO2 (see also chapter 8.2.1, 8.5.1, 8.8.1).
From day 50 on until the end of the test, only the following test replicates were sampled:
-test replicates containing the test item dispersion Silastol H 200 (10 mg organic C/L)
-test replicates containing the test item Silastol H 200-TS treated in ultrasonic bath (10 mg organic C/L)
-test replicates containing the test item dispersion Silastol H 200 (20 mg organic C/L) and aniline
On day 77, 5 mL HCl 2 M were added to each test flask in order to drive off dissolved CO2. On day 78, samples from both scrubber flasks from all test replicates were taken.
Additionally, at the start and at the end of the test 1 replicate of each the blank control and test item solution (for each test series) was taken and its TOC (unfiltered solution) and DOC (membrane filtered solutions, nylon filters 0.45 μm) was determined.
CO2 Determination
Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured in duplicate or triplicate, respectively (depending on the variation between the measured values). The carbon ana-lyser was calibrated with freshly prepared reference solutions containing potassium hydro-gen phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC) every month. After every start, quality control samples were measured.
Reference substance:
aniline
Key result
Parameter:
% degradation (CO2 evolution)
Value:
10
Sampling time:
77 d
Remarks on result:
other: First test series
Key result
Parameter:
% degradation (CO2 evolution)
Value:
16
Sampling time:
77 d
Remarks on result:
other: Second test series
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
77 d
Remarks on result:
other: Third test series

Degradation Values

In the following table, the percentage biodegradation is presented:

Day

Posi­

Posi­

Posi­

Test1

Test2

Test

Test1

Test2

Test

 

tive

tive

tive

10

10

Mean

20

20

Mean

 

Con­

Con­

Con­

mg/L

mg/L

10

mg/L

mg/L

20

 

trol1

trol2

trol

 

 

mg/L

 

 

mg/L

 

 

 

Mean

 

 

 

 

 

 

2

0.4

-0.6

-0.1

-1.0

0.2

-0.4

4.1

-2.3

0.9

4

6.4

4.8

5.6

1.6

2.8

2.2

5.2

-3.9

0.7

7

20.0

32.4

26.2

-1.3

1.9

0.3

5.6

-6.1

-0.2

9

51.9

56.9

54.4

2.0

5.1

3.6

7.4

-7.1

0.2

11

61.2

61.1

61.1

1.8

2.9

2.3

7.5

-8.5

-0.5

14

73.9

68.7

71.3

2.4

2.3

2.4

7.0

-9.3

-1.2

18

78.5

74.2

76.3

2.7

0.8

1.8

5.9

-9.9

-2.0

23

85.4

81.7

83.5

2.8

2.0

2.4

6.3

-8.8

-1.2

29

80.0

81.1

80.6

18.7

6.4

12.6

5.3

-6.5

-0.6

37

88.8

84.0

86.4

8.3

6.4

7.3

5.8

-4.5

0.7

44

85.6

79.7

82.7

8.9

7.7

8.3

5.6

-2.7

1.5

78

94.8

86.7

90.7

19.8

14.1

17.0

10.6

8.4

9.5

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The potential for biodegradation of the test item depends on the added amount of test item. At the organic carbon concentration of 10 mg/L a higher degree of biodegradation could be observed in all test series than at a concentration of 20 mg/L.
Further it could be observed that degradation was the highest at 10 mg/L of test item dis-persion Silastol H 200 (i.e. third test series), whereas it was lowest with (pure) Silastol H 200-TS when dissolved with methanol (i.e. first test series) but improved with (pure) Silas-tol H 200-TS when a homogeneous dispersion was produced by an ultrasonic bath (i.e. second test series).
Appropriate dispersion of the test item may therefore facilitate aerobic biodegradation up to a level for “ready biodegradability”.
Executive summary:

The study was performed to examine the potential for aerobic biodegradation of the test item. As is already known from other studies with the test item the biodegradability is as-sumed to highly depend on its bioavailability, particularly as the water solubility of the test item is low. In this study a series of 3 tests were performed with different methods for in-serting the test item into the test system for aerobic degradation.

In the first test series the test item (pure) Silastol H 200-TS was tested using a concentra-tion of nominal 10 mg organic carbon/L and 20 mg organic carbon/L respectively. To achieve a maximum bioavailability the test substance was dispersed as a thin layer on the surface of the test vessels: the nominal load of the test item (pure) Silastol H 200-TS was added into glass beakers and dissolved in a minimum amount of methanol. This solution was transferred into 2000 mL-SCHOTT-flasks. The glass beaker was rinsed twice with 5 mL methanol to guarantee complete transfer of test item into the test vessel. Then, metha-nol was evaporated completely using compressed air. Thus, a thin layer of test item on the surface of the test vessels was achieved.

In the second test series the test item (pure) Silastol H 200-TS was also tested using a concentration of nominal 10 mg organic carbon/L and 20 mg organic carbon/L respective-ly. The test item Silastol H 200-TS was homogenised in a glass beaker filled with an ali-quot of CO2-free test medium (10 mL) using an ultrasonic bath until a homogeneous dis-persion was reached and transferred into the test vessels. The beaker was rinsed three times with 10 mL CO2-free test medium.

In the third test series the test item dispersion Silastol H 200 was tested using a concentra-tion of nominal 10 mg organic carbon/L and 20 mg organic carbon/L respectively. The test item dispersion Silastol H 200 was weighed directly into the test flasks according the amount of organic carbon.

Because in the testing with 20 mg organic carbon/L in the third test series no degradation was observed on day 50, aniline was added to check for toxicity against the inoculum. A rapid increase of degradation was observed, thus the test item dispersion Silastol H 200 can be considered as not toxic toward the inoculum in a concentration of 72.8 mg/L.

The highest degradation was observed in the third test series at the carbon concentration of 10 mg/L. Test replicate 1 surpassed the pass level of 60 % degradation at the end of the test (63.3 %) whereas degradation of replicate 2 only reached 28.2 %. According to the guideline, the difference within the test replicates must be ≤ 20%. In this test series the difference was 31.5 %.

However, this test series was conducted to further characterise aerobic biodegradation of the substance, which is difficult to test because of its physico-chemical behaviour.

The aim of the study was to better describe the potential for aerobic biodegradation for the test item, achieve for comparability with test results from previous studies and testing of different methods for addition of the test item into the test system.

Therefore in this context, the validity criterion was not relevant for the acceptability of this study.

At the start and at the end of the test 1 replicate of each the blank control and test item solution (of each test series) was taken and its TOC (unfiltered solution) and DOC (membrane filtered solutions, nylon filters 0.45 μm) was determined. The measured TOC concentration at the start, especially in the second and third test series, proved the presence of test item in the test solution. At the end of the tests a clear decrease of the measured TOC concentrations in the second and third test series could be observed which confirmed the potential for biodegradation of the test substance.

Description of key information

In a study conducted according to OECD guideline 301F the test substance Silastol H 200 was proven to be not readily biodegradable; 0% degradation was observed, determined by oxygen consumption.

In a study conducted according to OECD guideline 301B the test substance Silastol H 200 TS was proven to be not readily biodegradable.

Since the degradation rate missed 60 % in the course of the test, Silastol H 200-TS is considered not to be readily biodegradable.

The potential for biodegradation of the test item depends on the added amount of test item. With an organic carbon concentration of 10 mg/L a higher degree of biodegradation could be observed than with 20 mg/L.

At the start and at the end of the tests 1 replicate of each the blank control and test item solution (for each test series) was taken and its TOC (unfiltered solution) and DOC (membrane filtered solutions, nylon filters 0.45 μm) was determined. The measured TOC concentrations at the start, especially in the second and third test series, proved the presence of test item in the test solutions. At the end of the tests a clear decrease of the measured TOC concentrations in the second and third test series could be observed which confirmed the potential for biodegradation of the test substance.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria
Type of water:
freshwater

Additional information

In a study conducted according to OECD guideline 301F the test substance Silastol H 200 was proven to be not readily biodegradable; 0% degradation was observed, determined by oxygen consumption.

In a study conducted according to OECD guideline 301B the test substance Silastol H 200 TS was proven to be not readily biodegradable.

The potential for biodegradation of the test item depends on the added amount of test item. With an organic carbon concentration of 10 mg/L a higher degree of biodegradation could be observed than with 20 mg/L.

At the start and at the end of the tests 1 replicate of each the blank control and test item solution (for each test series) was taken and its TOC (unfiltered solution) and DOC (mem-brane filtered solutions, nylon filters 0.45 μm) was determined. The measured TOC concentrations at the start, especially in the second and third test series, proved the presence of test item in the test solutions. At the end of the tests a clear decrease of the measured TOC concentrations in the second and third test series could be observed which confirmed the potential for biodegradation of the test substance.

However, there are reasonable doubts that the used methods are not suitable to evaluate the biodegradation potential of 1-Octadecanol, phosphate, potassium salt.

In a FDA Decision for food contact notification a mixture containing potassium alkyl phosphate salts, was considered to be easily biodegraded by non-adapted bacteria. In a Zahn-Wellens test, the bioelemination was shown to be 96 % after 3 days and 97 % after 6 days. In conclusion 95 % biodegradability as a conservative estimate was used for assessment by FDA.

M.C.Biotec Inc. reported Potassium Cetyl Phosphate to be not readily biodegradable in a Manometric Respirometry Test according to OECD guideline 301 F and to be inherently biodegradable in a MITI Test II according to OECD guideline 302 C. 

Furthermore the following information on readily biodegradability is published on the ECHA website. Phosphoric acid, mono-and di-C6 -10 alky esters were proven to be readily biodegradable in a screening test according to OECD guideline 301 B (Determination of the ready Biodegradability – Carbon Dioxide Evolution Test). As well the substance Phosphoric acid, 2-ethylhexyl ester is reported to be readily biodegradable, using the same test method.

Even though biodegradation of the substance under investigation could not be proven up to now, due to the structural similarity of the substances mentioned above biodegradation for 1-Octadecanol, phosphate, potassium salt is considered to be biodegradable under environmental condition.

 

Literature:

FDA, Environmental Decision Memo for Food Contact Notification No. 000388, April 2004, Environmental Toxicologist, Environmental Review Group (ERG)

MSDS, Potassium Cetyl Phosphate, M.C.Biotec Inc. No.40 Ma Jia Street, Nanjing 210009, China

 

Justification for read-across:

The main compounds of Phosphoric acid, C16-18-alkyl esters, potassium salts and 1-Octadecanol, phosphate, potassium salt are the mono and di-phosphoric esters.

The typical proportion of mono- to di-ester is 35 % to 50 % for both substances, respectively.

The only difference between the substances is varying chain lengths of the alkyl substituents, C16 and C18 or only C18.

Both substances are surfactants and have a polar “head” (the negatively charged phosphoric acid group) and a relatively inert hydrophobic “tail” (the long alkyl substituents).

The chemical behavior of both alkyl esters is expected to be very similar. The difference chain length of the alkyl substituent (C16 and C18 or only C18) is considered not relevant for the toxicological profile of the substances. This is proven by the close similarity of physico-chemical properties and the similar toxicological profile of the substances. 

In conclusion read-across for toxicological and eco-toxicological endpoints is considered valid without restrictions.