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Environmental fate & pathways

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:
28 Feb 2018 - 14 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a methanol-based test solution.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
Principles of method if other than guideline:
Three experiments were performed. Experiment 1 did not meet the validity criteria and was therefore disregarded. In Experiment 2, the results for the test item vessels were deemed unreliable since both test item vessels showed biodegradation significantly higher than 100%. Experiment 3 was performed to confirm the findings of the second experiment.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: freshly obtained sludge was kept under continuous aeration until further treatment.
- Pretreatment: The sludge was coarsly sieved (1 mm) and washed with mineral medium. After treatment the concentration of suspended solids (SS) was determined to be 2 g/L (in Experiment 2) or 3 g/L (in Experiment 3) in the concentrated sludge as used for the test. The sludge was used as inoculum at a concentration of 1 mL per litre of mineral medium, leading to a final concentration of SS of 2 mg/L (in Experiment 2) or 3 mg/L (in Experiment 3)
Duration of test (contact time):
28 d
Initial conc.:
28 mg/L
Based on:
test mat.
Remarks:
Experiment 2
Initial conc.:
64 mg/L
Based on:
ThOD
Remarks:
Experiment 2
Initial conc.:
36 mg/L
Based on:
test mat.
Remarks:
Experiment 3
Initial conc.:
81 mg/L
Based on:
ThOD
Remarks:
Experiment 3
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
See 'Any other information on materials and methods' for details on the set-up of test vessels.

TEST CONDITIONS
- Composition of medium: Mineral medium, according to OECD301
- Test temperature: 22 - 23 °C (Experiment 2); 22 - 24°C (Experiment 3).
- pH:
* Experiment 2: 7.9 - 8.1 at start, 7.6 - 7.8 at day 28
* Experiment 3: 7.8 - 8.2 at start, 7.5 - 7.8 at day 28
- pH adjusted: no
- Continuous aeration: yes
- Continuous darkness: yes
- Other: The test solutions were continuously stirred during the test to ensure optimal contact between the test item and test organisms.

TEST SYSTEM
- Culturing apparatus: 500 mL brown colored glass bottles, fill volume 244 or 360 mL, in order to obtain the required headspace-to-volume ratio.
- Number of culture flasks/concentration: 2 containing test item and inoculum; 2 containing only inoculum (inoculum blank); 1 containing reference item and inoculum (procedure control); 1 containing test item, reference item and inoculum (toxicity control).
- Preparation of bottles: At the start of the test (day 0) mineral medium was inoculated with activated sludge. Test and/or reference item were added to the respective bottles. The volumes of suspensions were made up to 244 or 360 mL using inoculated mineral medium. Rubber gaskets containing two pellets of sodium hydroxide were inserted into each bottle. Subsequently, each bottle was sealed by screwing an automated respirometer head on top.
- Method used to create aerobic conditions: continuous aeration
- Respirometer/measuring equipment: Lovibond BD600-GLP manometric respirometry system, equipped with an inductive stirring system.
- Details of trap for CO2: The CO2 produced in each test bottle reacted with the CO2 absorbent (Sodium hydroxide pellets) in the rubber gaskets. As gaseous O2 was converted into gaseous CO2 that was absorbed, the gas pressure in the test system slowly decreased. This decrease in air pressure was measured by the respirometer heads and automatically converted into oxygen consumption (mg O2/L).

MEASUREMENTS
- Measurements were recorded on day 0-4-7-12-14-18-21-25-28 (Experiment 2) or day 0-3-7-11-14-18-21-25-28 (Experiment 3). After recording the oxygen consumption on day 28, the pH was determined in all test vessels.
- Theoretical Oxygen Demand: The ThOD was calculated from the molecular formula.

CONTROL AND BLANK SYSTEM
- Abiotic sterile control: no
Reference substance:
other: 1-octanol
Preliminary study:
The first experiment was terminated after 14 days because oxygen consumption in the blanks was too high, breaching the acceptability criterion.
Parameter:
% degradation (O2 consumption)
Value:
123
Sampling time:
28 d
Remarks on result:
other: Experiment 2; Mean of two test vessels: 116% and 130%.
Key result
Parameter:
% degradation (O2 consumption)
Value:
95
Sampling time:
28 d
Remarks on result:
other: Experiment 3; Both test vessels showed 95% degradation
Details on results:
EXPERIMENT 2
- The relative biodegradation values calculated from the measurements performed during the test period revealed 130% and 116% biodegradation of test substance, for Bottles A and B, respectively (based on ThOD). Furthermore, average biodegradation of at least 60% was reached within a 10-day window. However, since a biodegradation significantly higher than 100% was observed, a third experiment was carried out.
- In the toxicity control, more than 25% biodegradation occurred within 14 days (85%, based on ThOD). Therefore, the test item was assumed not to inhibit microbial activity.

EXPERIMENT 3:
- The relative biodegradation values calculated from the measurements performed during the test period revealed 95% biodegradation of test substance, for both replicates (based on ThOD). Furthermore, average biodegradation of at least 60% was reached within a 10-day window.
- In the toxicity control, less than 25% biodegradation occurred within 14 days (9%, based on ThOD). However, in the second experiment the toxicity control reached 85% biodegradation. The results of the toxicity control in the second experiment are in line with what can be expected based on the biodegradation profile of the test item. Therefore, it was concluded the test item does not inhibit microbial activity.
Results with reference substance:
Functioning of the test system was checked by testing the reference item 1-octanol, which showed a normal biodegradation curve and 66% and 88% degradation within 14 days in Experiment 2 and Experiment 3, respectively.

Table 1: Comparison of Biodegradation of the Test Item in Bottles A and B– Experiment 2

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B1)

0

0

0

0

0

4

49

27

38

22

7

101

39

70

61

12

119

101

110

19

14

123

107

115

16

18

126

110

118

16

21

128

112

120

16

25

129

115

122

14

28

130

116

123

14

1): Absolute difference in biodegradation between bottles A and B

 

Table 2: Comparison of Biodegradation of the Test Item in Bottles A and B– Experiment 3

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B1)

0

0

0

0

0

3

14

14

14

0

7

30

77

53

47

11

45

91

68

46

14

69

97

83

29

18

84

98

91

14

21

97

97

97

0

25

97

97

97

0

28

95

95

95

0

1): Absolute difference in biodegradation between bottles A and B

Validity criteria fulfilled:
yes
Remarks:
See 'overall remarks' for details on validity criteria
Interpretation of results:
readily biodegradable
Conclusions:
The test substance was found to be readily biodegradable in two manometric respirometry tests.
Executive summary:

In a GLP-compliant Manometric Respirometry Test according to OECD guideline 301 F, the substance was assessed for its ready biodegradability. The test was performed three times: the first experiment was invalid and because the second experiment showed >100% biodegradation, a third experiment was performed to confirm the findings of the second experiment.

The substance was tested in duplicate at a target concentration of 28 and 36 mg/L, corresponding to a ThOD of 64 and 81 mg O2/L, in Experiment 2 and 3 respectively. The exposure period was 28 days and two inoculum blanks, a procedure control and a toxicity control were included. The substance was added directly into the test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the substance and test organisms.

In Experiment 2, 130% and 116% biodegradation was observed in two replicate bottles. In Experiment 3, 95% biodegradation was observed in both replicate bottles. Average biodegradation of at least 60% was reached within a 10 -day window in both experiments. The toxicity control reached 85% biodegradation within 14 days in Experiment 2, and less than 25% in Experiment 3. The results of the toxicity control in the second experiment are in line with what can be expected based on the biodegradation profile of the test item. Therefore, the test item was assumed not to inhibit microbial activity.

The criterion for ready biodegradability (at least 60% biodegradation within a 10 -day window) was met. Based on the outcome of the two experiments, the substance is considered to be readily biodegradable in the Manometric Respirometry Test. All validity criteria were met, thus the study was considered to be valid and reliable without restriction.

Description of key information

The test substance was found to be readily biodegradable in two manometric respirometry tests.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

In a GLP-compliant Manometric Respirometry Test according to OECD guideline 301 F, the substance was assessed for its ready biodegradability. The test was performed three times: the first experiment was invalid and because the second experiment showed >100% biodegradation, a third experiment was performed to confirm the findings of the second experiment.

The substance was tested in duplicate at a target concentration of 28 and 36 mg/L, corresponding to a ThOD of 64 and 81 mg O2/L, in Experiment 2 and 3 respectively. The exposure period was 28 days and two inoculum blanks, a procedure control and a toxicity control were included. The substance was added directly into the test bottles. The test solutions were continuously stirred during the test to ensure optimal contact between the substance and test organisms.

In Experiment 2, 130% and 116% biodegradation was observed in two replicate bottles. In Experiment 3, 95% biodegradation was observed in both replicate bottles. Average biodegradation of at least 60% was reached within a 10 -day window in both experiments. The toxicity control reached 85% biodegradation within 14 days in Experiment 2, and less than 25% in Experiment 3. The results of the toxicity control in the second experiment are in line with what can be expected based on the biodegradation profile of the test item. Therefore, the test item was assumed not to inhibit microbial activity.

The criterion for ready biodegradability (at least 60% biodegradation within a 10 -day window) was met. Based on the outcome of the two experiments, the substance is considered to be readily biodegradable in the Manometric Respirometry Test. All validity criteria were met, thus the study was considered to be valid and reliable without restriction.