Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-03-06 to 2018-05-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
26.09.2014
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.29 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test))
Version / remarks:
24 January 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): from the aeration stage of the local wastewater treatment plant, ARA Birs (Birsfelden / Switzerland), which treats predominantly domestic sewage. No pre-adaptation of the inoculum to the test item was done.

- Preparation of inoculum for exposure: The aerobic activated sewage sludge was washed three times by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in tap water and finally in mineral medium. Aliquots of the homogenized final sludge suspension were weighed, thereafter dried and the dry weight of the suspended solids was determined. Based on this determination, calculated amounts of wet sludge were suspended in mineral medium to obtain a concentration equivalent to 4 g dry material per liter. During the holding period of one day prior to use, the sludge was aerated with CO2-free air at room temperature. Prior to use, the dry weight of the sludge was again determined and the sludge was diluted with mineral medium to a concentration of 1 g dry material per liter. Defined volumes of the diluted activated sludge were added to the mineral medium in the test vessels to obtain a final concentration of 4 mg dry material per liter.
- Concentration of sludge: final concentration in the test vessels of 4 mg dry material per liter
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
24.6 mg/L
Based on:
test mat.
Initial conc.:
19.7 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral medium was prepared according to the testing guidelines and is considered not to contain any contaminant that would affect the integrity or outcome of the study. Analytical grade salts were dissolved in ultrapure water to obtain the stock solutions. To obtain the final mineral medium, 10 mL of stock solution No. 1 and 1 mL each of stock solutions Nos. 2, 3 and 4 were added to approximately 800 mL ultrapure water, mixed and made up to 1000 mL with ultrapure water. The pH was adjusted from 7.7 to 7.4 with a diluted hydrochloric acid solution.
- Additional substrate: no
- Solubilising agent (type and concentration if used): no
- Test temperature: 20 °C, in a temperature-controlled environment.
- pH adjusted: yes only in the mineral medium
- Aeration of dilution water: The sealed bottles were placed on an orbital shaker, with a shaking rate of 200 rpm, sufficient to keep the bottle contents well mixed and in suspension. The shaker was placed in a temperature-controlled environment.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The test was performed in “125 mL” Wheaton glass serum bottles, with a total volume of around 160 mL, sealed with aluminium crimp caps with Teflon lined chlorobutyl septa. 120 mL medium were added into each bottle, resulting in a headspace to liquid ratio of 1:3.
- Number of culture flasks/concentration: A sufficient number of bottles were prepared for each series to allow at least triplicate bottles to be measured on each sampling occasion. For the last sampling, five bottles (instead of triplicate bottles) from sets a), b) and c) were analyzed to enable 95 % confidence intervals to be calculated for the mean percentage biodegradation value.
The following number of bottles was prepared for each series (including bottles on reserve):
a) 35 bottles for the test item biodegradation, containing the test item in inoculated mineral medium,
b) 35 bottles for the inoculum control, containing inoculated mineral medium alone (blank),
c) 20 bottles for the procedure control, containing the reference item in inoculated mineral medium,
d) 15 vessels for the toxicity control, containing both, the test item and the reference item in inoculated mineral medium.
- Method used to create aerobic conditions/Test performed in closed vessels due to significant volatility of test substance:
Immediately before use, sufficient inoculated mineral medium was prepared by adding diluted activated sludge to mineral medium. Aliquots of inoculated medium (well mixed) were dispensed into replicate bottles to give a headspace to liquid ratio of 1:3 (i.e. 120 mL medium to 160 mL-capacity bottles). 3.7 μL of test item were injected into each sealed bottle containing the inoculated mineral medium using a high precision syringe. For the reference control 3.9 μL of 1-octanol were applied per bottle. The blank bottles were treated in a similar fashion but excluding test item and reference item. All bottles were sealed with Teflon-lined chlorobutyl septa and aluminum caps and incubated in the dark at 20 °C on a rotary shaker.

- Measuring equipment: The IC analyses were performed using the TOC infrared gas analyzer vario TOC cube from Elementar Analysensysteme GmbH, Germany, equipped with an automatic sampler. The IC analyzer was calibrated using Na2CO3 standard solutions in the range 0 to 30 mg IC/L. A standard sample was measured at each sampling occasion to prove conformity with the calibration curve.
- Other: CO2 production in the bottles was determined by measuring the increase in the concentration of inorganic carbon (IC) during incubation. By the addition of alkali and shaking, complete CO2 was converted to carbonate and the concentration of IC in the headspace was negligible.
Samples for analysis were withdrawn from the liquid phase. This was done as follows: an aliquot (1 mL) of a 7M NaOH solution was injected through the septum of each bottle sampled and the bottles were shaken for 1 h at the test temperature. The same NaOH solution was used for all bottles sacrificed on a particular day. As a control the IC contribution of the used NaOH solution was also determined. The test bottles were removed from the shaker and allowed to settle. Suitable volumes of the liquid phase were withdrawn and subjected to IC analysis.

SAMPLING
- Sampling frequency: Bottles were sacrificed for analysis on the following sampling days:
Test item and inoculum control: Exposure Day 0, 2, 5, 7, 9, 14, 21 and 28,
Procedure control: Exposure Day 0, 2, 7, 14 and 28,
Toxicity control: Exposure Day 0, 7, 14 and 28.
- Sampling method: Triplicate bottles were measured on each sampling occasion throughout the test. However, five bottles from each series (except the toxicity control) were analyzed at the end of the test to enable 95 % confidence intervals to be calculated for the mean percentage biodegradation value.
- Other: In addition, the pH was measured in a test item and an inoculum control bottle (which have not been made alkaline) at the start and at the end of incubation.

CONTROL AND BLANK SYSTEM
- Inoculum blank: containing inoculated mineral medium alone (35 bottles)
- Abiotic sterile control: none
- Toxicity control: containing both, the test item and the reference item in inoculated mineral medium (15 vessels)
- Procedure control, containing the reference item in inoculated mineral medium (20 bottles)
Reference substance
Reference substance:
other: 1-octanol

Results and discussion

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
3.6
Sampling time:
28 d
Remarks on result:
other: 95% CI: +/- 2.0%
Details on results:
Biodegradation in the Toxicity Control:
The percent biodegradation in the toxicity control was calculated to be 77 % by Exposure Day 7 and 92 % at the end of the 28-day exposure period. To assess if the test item may had inhibitory effect on the activity of the inoculum the following calculation formula was applied for the Day 28: (%degradation reference item − %degradation toxicitycontrol)/%degradation reference item x 100
The resulting value was – 93 %, which is lower than 25 %. Thus, according to the test guidelines, the test item Tri-n-hexylamine had no inhibitory effect on activated sludge microorganisms at the tested concentration of 24.6 mg/L.
Total Inorganic Carbon in the Inoculum Control:
The mean concentration of total inorganic carbon (TIC) in the inoculum control on Day 28 was 2.4 mg C/L. Therefore, the test is valid because the mean concentration of TIC in the inoculum control at the end of the test was < 3 mg C/L.
pH Measurement:
The pH measured in a test item and an inoculum control bottle (which have not been made alkaline) at the start of incubation was 7.3 and 7.4, respectively. The measurements at the end of the test, on Day 28 resulted in pH 7.9 and 7.3, respectively.

BOD5 / COD results

Results with reference substance:
In the procedure control, the reference item 1-octanol was degraded by an average of 78 % and 74 % by Exposure Day 7 and 14, respectively, thus confirming suitability of the activated sludge (> 60 % degradation by Exposure Day 14). At the end of the exposure period, on Day 28, the mean percentage biodegradation of the five procedure control bottles sampled was 48 % with a 95 % confidence interval of ± 27 %.

Any other information on results incl. tables

Table 1     Inorganic Carbon Concentration (IC) in the Test Flasks

 

 

IC found in test flasks (IC in mg C/L)

Time [days]

Test item1

Reference item1

Replicate No.

Replicate No.

1

2

3

4

5

mean

1

2

3

4

5

mean

0

-0.2

-0.5

-0.1

--

--

-0.3

-0.2

0.2

0.0

--

--

0.0

2

0.1

0.1

0.2

--

--

0.1

6.2

6.7

5.8

--

--

6.2

5

-0.1

-0.8

1.6

--

--

0.2

--

--

--

--

--

n.a.

7

0.3

1.6

-0.4

--

--

0.5

17.5

17.1

11.6

--

--

15.4

9

1.2

0.3

0.5

--

--

0.7

--

--

--

--

--

n.a.

14

-0.4

-0.3

0.0

--

--

-0.2

17.1

8.6

18.0

--

--

14.6

21

0.0

-0.3

0.5

--

--

0.1

--

--

--

--

--

n.a.

28

0.4

0.9

1.3

1.0

0.2

0.7

2.9

6.5

12.9

7.0

18.2

9.5

 (table continued below)

 

 

 

IC found in test flasks (IC in mg C/L)

Time [days]

Inoculum control

Toxicity control1

Replicate No.

Replicate No.

1

2

3

4

5

mean

1

2

3

mean

0

2.0

1.7

2.6

--

--

2.1

1.6

1.7

0.9

1.4

2

1.4

1.4

1.1

--

--

1.3

--

--

--

n.a.

5

2.6

2.2

2.7

--

--

2.5

--

--

--

n.a.

7

2.0

1.7

2.5

--

--

2.1

16.4

14.6

14.9

15.3

9

1.9

1.9

2.1

--

--

2.0

--

--

--

n.a.

14

2.7

1.9

1.9

--

--

2.2

16.8

15.1

14.6

15.5

21

1.3

2.1

2.3

--

--

1.9

--

--

--

n.a.

28

3.0

3.0

2.2

1.9

2.1

2.4

20.8

18.8

15.2

18.2

 

1:      Corrected for the mean inoculum control.

--:     No samples taken.

n.a.:  Not applicable.

 

  

Table 2     Percentage Biodegradation of the Test Item Tri-n-hexylamine and the Reference Item 1-Octanol during the Incubation Period

 

 

% Degradation1

Time [days]

Test item

Reference item

Replicate No.

Replicate No.

1

2

3

4

5

mean

1

2

3

4

5

mean

0

-1.0

-2.5

-0.5

--

--

-1.4

-1.0

1.0

0.0

--

--

0.0

2

0.5

0.5

1.0

--

--

0.7

31.3

33.8

29.2

--

--

31.4

5

-0.5

-4.1

8.1

--

--

1.2

--

--

--

--

--

n.a.

7

1.7

8.3

-1.9

--

--

2.7

88.4

86.4

58.7

--

--

77.8

9

6.2

1.7

2.7

--

--

3.5

--

--

--

--

--

n.a.

14

-1.9

-1.4

0.2

--

--

-1.0

86.4

43.5

90.9

--

--

73.6

21

0.0

-1.5

2.5

--

--

0.3

--

--

--

--

--

n.a.

28

1.8

4.4

6.4

4.9

0.8

3.6

14.4

32.6

64.8

35.1

91.6

47.7

 (table continued below)

 

 

 

% Degradation

Time [days]

Toxicity control2

Replicate No.

1

2

3

mean

0

8.1

8.6

4.5

7.1

2

--

--

--

n.a.

5

--

--

--

n.a.

7

82.9

73.8

75.3

77.3

9

--

--

--

n.a.

14

84.9

76.3

73.8

78.3

21

--

--

--

n.a.

28

104.7

94.6

76.4

91.9

 

1:      Negative values due to a higher IC formation in the inoculum control.

2:      Based on the added TOC from the reference item only.

--:     No samples taken.

n.a.:  Not applicable.

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See above remarks
Interpretation of results:
not readily biodegradable
Conclusions:
The test item Tri-n-hexylamine was found to be not biodegradable after 28 days of exposure to activated sludge under the conditions of the conducted CO2 in Sealed Vessels Test (Headspace Test).
Executive summary:

The test item Tri-n-hexylamine was investigated for its ready biodegradability in a Headspace Test (CO2 in sealed vessels) over 28 days according to the OECD Guideline for Testing of Chemicals, No. 310 (2014) and the Method C.29 of Commission Regulation (EU) No 260/2014 and following GLP.

The test was performed, at a test item concentration of 24.6 mg/L, corresponding to 19.7 mg C/L and in the presence of activated sludge as inoculum at the final concentration of 4 mg dry material per liter mineral medium. The sealed glass serum bottles with a headspace to liquid ratio of 1:3 where incubated at 20 °C in the dark whit a shaking rate of 200 rpm. The inorganic carbon (IC) production in the test bottles containing Tri-n-hexylamine in inoculated mineral medium was in the range of the inoculum controls throughout the exposure period of 28 days. The calculated maximal mean level of biodegradation, i.e. 3.6 %, was reached at the Exposure Day 28. Consequently, the test item Tri-n-hexylamine was not biodegradable under the test conditions within 28 days.

In the procedure control, the reference item 1-octanol was degraded by an average of 78 % and 74 % by Exposure Day 7 and 14, respectively, thus confirming suitability of the activated sludge (> 60 % degradation by Exposure Day 14).

In the toxicity control, containing both the test item and the reference item, no inhibitory effect on the biodegradation of the reference item was determined. Thus, Tri-n-hexylamine had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 24.6 mg/L, corresponding to 19.7 mg C/L.

All the validity criteria of the test were met.