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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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Qualifier:
according to guideline
Guideline:
other: Method for Testing the Biodegradability of Chemicals Substances by microorganisms, Japan
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: On-site sludge sampling was carried out at the following 10 locations in Japan; sampling date was in June 1993; city sewage was sampled in return sludge from sewage plants and from lakes, rivers and sea surface water and soil was collected if was in contact with atmosphere
Fushikogawa city sewage plant (Sapporo-shi, Hokkaido)
Fukashiba industrial sewage plant (Kashima-gun, Ibaraki)
Nakahama city sewage plant (Osaka-shi, Osaka)
Ochiai city sewage plant (Shinjuku-ku, Tokyo)
Kitakami River (Ishinomaki-shi, Miyagi)
Shinano River (Niigata-shi, Niigata)
Yoshino River (Tokushima-shi, Tokushima)
Lake Biwa (Otsu-shi, Shiga)
Hiroshima Bay (Hiroshima-shi, Hiroshima)
Dookai Bay (Kitakyushu-shi, Fukuoka)
- Preparation of inoculum: Activated sludge was prepared as follows to maintain its uniformity. The filtrate (5L) of the supernatant of the activated sludge, in use as present was mixed with 500mL of the filtrate of the supernatant of a newly collected sludge and the mixture was cultered at 7.0±1.0 under the suffucient aeration.
- Concentration of sludge: 4000 mg/L
- Method of cultivation: About 30 minutes after ceasing aeration of the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed. Dichlorinated water was added to the remaining portion so that the total volume reached 10L. This mixture was aerated, and then a predetermined amount of synthetic sewage was 0.1 wt% in the volume of dichlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 25±2 °C.
- composition of synthetic sewage: glucose, peptone and monopotassium phosphate were dissolved in dechlorinated water. each concentration was 5 (W/V)% and the solution was adjusted to pH 7.0±1.0 with sodium hydroxide.
- Control and use of sludge: During cultivation, the appearance of the supernatant, setting of the sludge, formation of flock of flock, pH, dissolved oxygen concentration in the solution and temperature were checked and necessary adjustments were made. Microflora in the activated sludge was microscopically observed and the sludge with the no abnormal symptoms was used for the test.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Details on study design:
TEST CONDITIONS
- Composition of medium: Each 3 mL of solutions A, B, C and D, which are prescribed in JIS K 0102-1986-21, was made up to 1000 mL with purified water (Takasugi Seiyaku Co., Ltd.), and then the pH of this solution was adjusted to 7.0.
- Test temperature: 25 ± 1 °C
- pH adjusted: yes
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: Closed system oxygen consumption measuring apparatus (coulometer: Ohkura Electric Co., Ltd.); data processor (data sampler: Asahi Instrument Industries Co., Ltd.)
- Number of culture flasks/concentration: 3
- Measuring equipment: pH meter (Toa Electronics Ltd. type HM-50S); Electric reading balance (Satorius type 2007 MP6)
- Test performed in open system: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Other: control blank

STATISTICAL METHODS:
Reference substance:
aniline
Parameter:
other: BOD
Value:
43
Sampling time:
28 d
Parameter:
other: HPLC
Value:
74
Sampling time:
28 d
Details on results:
It was considered that the difference between the two values might be explained in terms of the decrease of the substance due to a physical-chemical mechanism or an adsorption to the sludge. Therefore, in order to confirm whether the test substance was adsorbed to the sludge or not, a separate experiment was carried out. Various amount of sludge was added to vessels containing the test substance and the basal culture medium, and the vessels were stirred for 24 hours. Determination of the test substance concentration in the solutions did not reveal any adsorption to the sludge.
Results with reference substance:
The percentage of aniline (calculated from the BOD values) was 70% and 79% on the 7th and 14th day, respectively. It was concluded that the conditions were valid.

Table 1: Appearances of the test solutions

 

Solution

Appearance

At the initiation of culturing

Water + test substance

Test substance was not dissolved.

Sludge + test substance

Test substance was not dissolved.

At the termination of culturing

Water + test substance

Test solution was white cloudy.

Sludge + test substance

Test solution was white cloudy and growth of the sludge was observed.

Table 2: The percentage biodegradation after 28 days.

Method

Percentage biodegradation

Vessel 1

Vessel 2

Vessel 3

Average

BOD

53

47

29

43

HPLC

93

82

46

74

Interpretation of results:
other: not readily biodegradable
Conclusions:
It was concluded that the test substance for the greater part was split into ethanol and a silanol, that the ethanol was completely degraded by microorganisms, and that the test substance as well as the silanol was partially degraded by microoganisms. A complete degradation, however, was not reached under the present test conditions.
Endpoint:
biodegradation in water: screening test, other
Remarks:
enhanced ready biodegradability test (60 d)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Apr - 13 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
60 days duration
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Hildesheim, Germany
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant, Hildesheim, Germany
-Pretreatment: activated sludge was washed twice with chlorine free tap water. Thereafter settled sludge was resuspended in mineral salts medium and was aerated for 2 h. Sludge was then homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with air for 5 d.
- Concentration of sludge: 10 mL/L were used to initiate inoculation
- Initial cell/biomass concentration: approx. 1.13 x 10^7 CFU/L in the final test solution
Duration of test (contact time):
60 d
Initial conc.:
19 mg/L
Based on:
test mat.
Initial conc.:
52.8 mg/L
Based on:
ThOD
Initial conc.:
2.78 other: mg O2/mg
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral salts medium according to OECD 301 F
- Test temperature: 20 - 21 °C
- pH: 7.60 - 7. 90
- pH adjusted: no
- Concentration of sludge: 10 mL/L were used to initiate inoculation
- Continuous darkness: yes
- Other: continuous stirring

TEST SYSTEM
- Number of culture flasks/concentration: 2
- Measuring equipment: OxiTop® measuring heads, WTW Stirring platform, WTW
- Test performed in closed vessels: yes
- Details of trap for CO2: a rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2
- Other: separate replicates of test item and toxicity control were prepared for measurement of pH at test start

SAMPLING
- Sampling frequency: 1080 measuring points during the 60 d incubation period

CONTROL AND BLANK SYSTEM
- Inoculum control: 2
- Inoculum control with silicone oil: 2 (500 µL silicone oil were pipetted directly into the inoculated test medium)
- Toxicity control: 1
- Functional control: 1

STATISTICAL METHODS: Excel, MICROSOFT CORPORATION, SigmaPlot, SPSS CORPORATION

Reference substance:
benzoic acid, sodium salt
Remarks:
45 mg/L
Parameter:
% degradation (O2 consumption)
Value:
57
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
60 d
Details on results:
- Reference substance: 100% degradation after 23 d (pass level of > 60% was reached after 3 d)
- Toxicity control: 75% degradation after 28 d (82% degradation after 60 d)

VALIDITY CRITERIA:

Table 1: Validity criteria

Criterionfrom the guideline

Outcome

Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

6% difference on day 28 and 13% on day 60

yes

Percentage degradation of the reference compound has reached the pass levels by day 14.

60% after 3 d

yes

The oxygen uptake of the inoculum blank is normally 20-30 mg O2/L and should not be greater than 60 mg/L in 28 days.

mean oxygen depletion in the inoculum control was 8 mg O2/L on day 28 and 10.8 mg O2/L on day 60

yes

 

 

 

RESULTS:

Both test item replicates reached the 10% biodegradation level on day 4. The 60% pass level was reached by the mean of replicates on day 39. The mean biodegradation on day 60 was 65%.

Table 2: % biodegradation over time.

[d] Functional Control Biodegradation [%] Toxicity Control
Test item
1 2
1 7 0 0 4
2 50 0 0 31
3 62 3 1 37
4 73 16 14 43
5 79 26 25 48
6 82 31 29 52
7 84 35 34 55
8 86 38 35 58
9 88 40 38 61
10 89 42 40 62
11 90 42 42 63
12 91 44 42 64
13 93 45 44 65
14 93 46 44 66
15 94 47 45 67
16 96 48 46 68
17 97 49 48 69
18 98 51 49 70
19 98 52 50 70
20 99 52 50 71
21 98 52 50 71
22 99 52 52 72
23 100 53 52 72
24 100 54 52 73
25 100 54 52 74
26 100 56 53 74
27 100 60 53 74
28 100 60 54 75
29 100 64 53 75
30 100 64 53 76
31 100 63 52 75
32 100 63 53 76
33 100 64 53 77
34 100 64 52 77
35 100 64 52 77
36 100 65 54 77
37 100 65 54 78
38 100 65 54 78
39 100 66 54 78
40 100 66 53 79
41 100 66 54 79
42 100 67 54 79
43 100 67 53 79
44 100 66 53 79
45 100 67 54 80
46 100 68 53 80
47 100 68 54 80
48 100 68 55 80
49 100 68 55 80
50 100 68 55 81
51 100 68 55 80
52 100 69 54 81
53 100 70 54 81
54 100 70 54 81
55 100 70 54 81
56 100 70 54 81
57 100 69 55 81
58 100 70 57 81
59 100 70 58 81
60 100 71 58 82
Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
inherently biodegradable
Conclusions:
A biodegradation study according to OECD 301F resulted in a biodegradation of 57% after 28 d and 65% after 60 d.
Endpoint:
biodegradation in water: screening test, other
Remarks:
enhanced ready biodegradability test (60 d)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the attached justification below and the overall justification for grouping of substances attached in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Parameter:
% degradation (O2 consumption)
Value:
57
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
65
Sampling time:
60 d
Details on results:
- Reference substance: 100% degradation after 23 d (pass level of > 60% was reached after 3 d)
- Toxicity control: 75% degradation after 28 d (82% degradation after 60 d)
Interpretation of results:
inherently biodegradable
Conclusions:
A biodegradation study according to OECD 301F resulted in a biodegradation of 57% after 28 d and 65% after 60 d.

Description of key information

Triethoxy(hexadecyl)silane (CAS 16415-13-7) is inherently biodegradable (57% after 28 d and 65% after 60 d, OECD 301F) based on read across from CAS 18643-08-8. 

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

One experimental study with triethoxy(hexadecyl)silane (CAS 16415-13-7) is available. The study was performed according to OECD 301 C (GLP). A mixture of activated sludge and samples from different natural locations (e.g. lakes, rivers) was used as inoculum. The test item was degraded to 43% (BOD analysis) and 74% (HPLC analysis). Based on the hydrolytic half-life of 58.4 h at pH7 the substance is expected to hydrolyse within the timescale of a ready biodegradation study. During hydrolysis ethanol and hexadecylsilanetriol are formed. It is expected that ethanol was completely degraded by microorganisms, and that the silanol hydrolysis product was partially degraded by microorganisms. A complete degradation, however, was not reached under the present test conditions.

In order to verify if the substance is at least inherently biodegradable and thus not persistent in the environment a read-across to the suitable source substance chloro(dimethyl)octadecylsilane (CAS 18643-08-8) has been applied to support the environmental hazard assessment of the substance. This read-across is in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5 “Grouping of substances and read across” and in accordance with the read across assessment framework (RAAF, ECHA 2017). Details on the read across justification can be found in the attached justification in the respective target entry and in the overall justification for grouping of substances attached in IUCLID Section 13.

The available study investigating the biodegradation of the analogue substance chloro(dimethyl)octadecylsilane (CAS 18643-08-8) was conducted according to OECD guidance 301F and GLP conditions. The inoculum (non-adapted activated sludge from a sewage treatment plant) was exposed for 60 days to 19 mg/L test substance and biodegradation was calculated based on CO2 evolution. The substance was degraded to 57% after 28 d and 65% after 60 d. Therefore, the read across substance is considered as inherently biodegradable.

Based on the result of the analogue substance, triethoxy(hexadecyl)silane (CAS 16415-13-7) is assumed to be inherently biodegradable.

[Type of water: freshwater]