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Environmental fate & pathways

Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: simulation testing on ultimate degradation in surface water
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
see attached justification
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Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): In March, June, September, and December, sludge was sampled at the following 10 places in Japan: 1. Fukogawa city sewage plant, 2. Fukashiba industry sewage plant, 3. Nakahama city sewage plant, 4. Ochiai city sewage plant, 5. Kitakami river, 6. Shinano river, 7. Yoshino river, 8. Lake Biwa, 9. Hiroshima bay, 10. Dookai bay; sampling: 1. City sewage: Returned sludge from sewage plants was taken. 2. Rivers, lake and sea: Surface water and surface soil which were in contact with atmosphere were collected.
- Method of cultivation: About 30 minutes after ceasing aeration to the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed. Then the equal volume of dechlorinated water was added to the remaining portion and aerated again, followed by addition of synthetic sewage at a concentration of 0.1% (w/v). This procedure was repeated once every day. The culturing was carried out at 25 ± 2 °C. 5 L of the filtrate of the supernatant of old activated sludge was mixed with 500 mL of the filtrate of the supernatant of new sludge and cultured at pH 7.0 ± 1.0 under sufficient aeration using prefiltered open air. During the cultivation, appearance of the supernatant, precipitability, formation 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 sludge with no abnormal symptom was used for the test.
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 3 mL each of four stock solutions, as described in JIS K 0102-1986-21, are diluted to 1000 mL with purified water
- pH: 7.0
- pH adjusted: yes
- Suspended solids concentration: determined according to Method Japanese Industrial Standards (JIS) K 0102-1986-14.1

TEST SYSTEM
- Culturing apparatus: Closed system oxygen consumption measuring apparatus (Coulometer: Ohkura Electric Co., Ltd.); 300 mL vessel, absorbent for evolving carbon dioxide Soda lime No .l (extra pure reagent, Wako Pure Chemical Industries, Ltd.).
- Number of culture flasks/concentration: 1
- Measuring equipment: Coulometer, Okhura Electric Co., Ltd.
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: soda lime, extra pure, Wako Pure Chemical Industries, Ltd.)

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: no
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
9 - 12
Sampling time:
28 d
Interpretation of results:
other: poorly biodegradable
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: aerobic activated sludge from a wastewater treatment treating predominantly domestic wastewater
- Preparation of inoculum for exposure: Sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was diluted with test water to a concentration of about 1 g dry material per liter. Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.

Duration of test (contact time):
28 d
Initial conc.:
33 mg/L
Based on:
test mat.
Initial conc.:
15.3 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: One day before test start (Day-1), between 2400 and 3000 mL of untreated test medium was filled into the flasks and 90 ml activated sludge inoculum was added. Then aeration overnight with C02-free air on the following day (Day 0), defined amounts of the test item were directly added to the test flasks and made up to a volume of 3 L with test water.
- Solubilising agent: none
- Test temperature: 21-23 °C
- pH: 7.2 - 7.4
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5-liter all-glass amber bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Air was led through a bottle containing about 750 ml of a 2 M NaOH solution to trap CO2. The C02-free air was passed through the test solutions at a rate corresponding to about 30-100 ml/min.
- Details of trap for CO2 and volatile organics if used: Two absorber flasks, the first one containing 300 ml 0.05 M NaOH and the second one
containing 200 ml 0.05 M NaOH, were connected in series to the exit air line of each test flask.

SAMPLING
- Sampling frequency:
- Test item and inoculum control: 2, 5, 7, 9, 12, 14, 19, 23, 27, 28, 29
- Procedure control: 2, 7, 14, 28, 29
- Toxicity control: 7, 14, 28, 29
- Sampling method: aliquot of 5.0 ml withdrawn from the absorber flask nearest to the test flask for analysis of inorganic carbon

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Toxicity control: 1 replicate
- Procedure control: 2 replicates



Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Results with reference substance:
Mean degradation rate after 28 days = 77.1%
Interpretation of results:
under test conditions no biodegradation observed
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sampled at 10 locations in Japan
- Concentration of sludge: 30 mg/l
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Test temperature: 25 ± 1°C


TEST SYSTEM
- Culturing apparatus: Asahi Techneion Co., Ltd.
- Number of culture flasks/concentration: 3


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes
Reference substance:
aniline
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Interpretation of results:
under test conditions no biodegradation observed
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1978
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Limited information available.
Qualifier:
according to
Guideline:
other: Notice No. 5 of the Environmental Health Affairs Division; Notice No. 615 of the Pharmaceutical Affairs Bureau Utilizing a test of degradation of a chemical substance by microorganisms; Notice No. 392 of the Basic Industries Bureau (Kikyoku No.392), 1974
GLP compliance:
no
Oxygen conditions:
aerobic
Details on inoculum:
Sludge density: 30 ppm
Duration of test (contact time):
2 wk
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Parameter:
% degradation (O2 consumption)
Value:
0
Details on results:
Results according to an absorption spectrophotometer: Negatives values were obtained.
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
additional information on environmental fate and behaviour
Remarks:
Dispersion stability in simulated environmental media
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 318
GLP compliance:
yes (incl. certificate)

At any of the time points mentioned in the TG-318, the influence of Ca is critical. Regardless of pH, the pigment is categorized at the 24h-sampling time as “instable” in 10 mM Ca, representing high water hardness. At 6h, most media induce “intermediate stability”, and only one medium (0 mM Ca, pH 9) induces a stability above 90%. At 24h, all media at 0 mM Ca and 1 mM Ca induce an intermediate stability between 60% and 87%. The stabilities systematically decrease over time, decrease slightly between 0 mM Ca and 1 mM Ca. The difference between pH 4 and pH 7 is low, but pH 9 systematically induces a slightly higher stability.

Table 1: Full results of the dispersion stability in the presence of NOM

Ca(NO3)2

Stability after 6h

Standard deviation

Stability after 15h

Standard deviation

Stability after 24h

Standard deviation

[mM]

[%]

[%]

[%]

[%]

[%]

[%]

pH 4

0

84.2

0.7

75.3

1.2

64.5

1.1

pH 4

1

81.9

0.4

70.3

0.7

60.3

0.6

pH 4

10

9.0

0.8

3.3

0.6

2.3

0.5

pH 7

0

80.3

0.6

70.6

0.3

63.4

0.6

pH 7

1

81.3

0.7

69.9

0.0

62.0

0.5

pH 7

10

9.7

0.9

4.1

0.2

3.2

0.2

pH 9

0

93.8

0.6

90.0

1.3

86.7

1.6

pH 9

1

86.9

0.5

75.2

1.8

69.3

1.3

pH 9

10

10.6

0.2

5.9

0.3

4.5

0.4

 

 

The results showed that under NOM-free conditions, the pigment was minimally less stable.The differences are not statistically significant.

 

Table 2: Comparison of the results of the dispersion stability with and without the presence of NOM

sedimentation time

6h

24h

1mM Ca, pH7, with NOM

99.45±0.39

99.22 ± 0.05

1mM Ca, pH7, without NOM

99.67 ± 0.02

98.81 ± 0.29

 

To rationalize the observed dispersion stability, the particle size distribution directly in the environmental medium (exact same sample preparation as for the UV/VIS measurements) was checked.

The NanoDefine method of Analytical Ultracentrifugation (SOP AUC-RI, published by 3) was applied. The centrifugation parameters are given above.

The observed size distributions confirm the low agglomeration at 0 mM and 1 mM Ca, against the high agglomeration at 10 mM Ca.

If the particles would have been significantly dissolved, no size distribution would be observable at all by this method, which relies on the detection of the movement of particles during centrifugal separation.

Additionally, the centrifugation methods include a determination of the remaining absorption after centrifugation, fully consistent with the conventional determination of the dissolved fraction after centrifugation as recommended by the TG-318. Even for the case of highest stability (pH 9, 0 mM Ca, with NOM), the remaining absorption was measured at 0.0504 ± 0.0024. This is a fraction of 2% of the initial absorption, but actually is close to the LOD of the built-in UV/Vis detector. Considering the LOD, between 0% and 2% of the sample may have been dissolved.

All evidence combined, the results after centrifugation confirm that at least 98% of the observed dispersion stability has to be attributed to the particles, not to dissolution.

Executive summary:

We found that the organic pigment is rather insensitive to pH changes, with little differences in dispersion stability between pH 4 and pH 9.

This is a significant difference against the metal oxide (TiO2) that was proposed as benchmark material of “intermediate stability” by the TG318.

Dissolution was excluded as the main cause of the apparent stability. The dispersion stability of Pigment Red 53:1 is of “intermediate stability” and depends especially on water hardness.

Only in very hard water with 10mM Ca, the stability of “low”. In all other conditions, the stability is at least “intermediate”, such that the aquatic compartment is relevant to risk assessment.

Data source

Materials and methods

Results and discussion

Transformation products:
no
Remarks:
not expected according to substance properties

Applicant's summary and conclusion