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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:
Experimental Starting Date: August 20, 2018 and Experimental Completion Date: September 21, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Version / remarks:
OECD No. 301 A (1992)
Commission Regulation (EC) No. 440/2008, C.4-A (2008)
OPPTS 835.3110, Paragraph (l) (1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-[1,3-phenylenebis(oxy)]bisethanol
EC Number:
203-028-3
EC Name:
2,2'-[1,3-phenylenebis(oxy)]bisethanol
Cas Number:
102-40-9
Molecular formula:
C10H14O4
IUPAC Name:
2-[3-(2-hydroxyethoxy)phenoxy]ethan-1-ol
Test material form:
other: crystal powder
Details on test material:
- Synonym (Trade Name): ADDITIVE® 9735
- CAS Number: 102-40-9
- Molecular Formula: C10H14O4
- Molecular Weight: 198 g/mol
- Appearance: White crystal powder
- Expiration date: 30 August 2019
- Purity: 100%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 17083101
- Expiration date of the lot/batch: August 30, 2019
- Purity: 100 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, at 20 ± 5 °C. Keep container tightly closed in a dry and well-ventilated place.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, 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.
- Concentration of sludge: a calculated amount of wet sludge was 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 at room temperature. Defined amounts of the activated sludge suspension were added to mineral medium to obtain a final concentration of 30 mg dry material per liter.
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
28.6 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium
KH2PO4: 8.50 g/L
K2HPO4: 21.75 g/L
Na2HPO4 × 2H2O: 33.40 g/L
NH4Cl: 0.50 g/L
The pH of this solution was 7.4.
CaCl2 × 2H2O: 36.40 g/L
MgSO4 × 7H2O: 22.50 g/L
FeCl3 × 6H2O: 0.25 g/L, stabilized with one drop of concentrated HCl per liter.

To obtain 1 L of 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.

- Test temperature: The test was conducted at 22 °C. The incubator temperature was recorded continuously.
- pH: The pH was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution.
- pH adjusted: yes, from 7.8 to 7.4 with a diluted hydrochloric acid solution.
- Light conditions: The test vessels were incubated in diffuse light.

TEST SYSTEM
- Culturing apparatus: 2000 mL Erlenmeyer glass vessels, cleaned with HCl, rinsed with ultrapure water and dried. The test vessels were filled to a volume of 1000 mL. The test vessels were labeled with the study number and all necessary information to ensure unique identification.
- Method used to create aerobic conditions: test vessels were incubated in an incubator (i.e. Multitron Pro from Infors AG, Bottmingen, Switzerland) with automatic temperature control and equipped with an orbital platform shaker, at approximately 130 rpm to prevent settlement of the inoculum and to maintain aerobic conditions.
- Number of culture flasks/concentration: 2
- Measuring equipment: DOC analyses were performed using a TOC infrared gas analyzer equipped with an automatic sampler (i.e. vario TOC cube from Elementar Analysensysteme GmbH, Langenselbold, Germany).
- Test performed in closed vessels due to significant volatility of test substance: Each vessel was loosely covered with an aluminum cap to reduce losses by evaporation.
- Test performed in open system: no

SAMPLING
- Sampling frequency: For the DOC analyses one sample of about 10-12 mL was taken from each sampled test vessel per sampling occassion. Prior to sampling, water evaporation losses were determined by weighing the vessels and were compensated by adding purified water. Deposits on the test vessels were resuspended. Sampling dates:
*Test item and inoculum control: Exposure Day 0, 3, 5, 7, 10, 12, 14, 18, 21 and 28
*Procedure control: Exposure Day 0, 3, 7, 14 and 28
*Toxicity control: Exposure Day 0, 7, 14 and 28

Sample method: Samples were filtered through a 0.45 µm filter. The first 2-3 mL of the filtrate were discarded. Thereafter, the filtrates were immediately analyzed for DOC. The filtrates of the samples taken at the Day 14 were stored frozen (-20±5 °C) for five days, since immediate analysis was not possible. After this storage period the filtrates were thawed at room temperature and analyzed as above described.
Reference substance
Reference substance:
other: sodium benzoate

Results and discussion

Preliminary study:
n/a
Test performance:
The results are considered to be valid since the following criteria are met:
- The percentage degradation of the reference item reached with 92 % by Exposure Day 3 the pass level for ready biodegradability (criterion: at least 70 % in a 10-day window by Day 14).
- Since the test item was not degraded, the validity criterion regarding thedifference between the DOC removal values of the replicate test item vessels isnot applicable.
-  The test item is considered to be non-inhibitory, since the toxicity control attained 52 % degradation by Day 14 (criterion: 35% degradation by Day 14).
% Degradation
Key result
Parameter:
% degradation (DOC removal)
Value:
9
Sampling time:
28 d
Details on results:
Biodegradation of the Test Item
In the test vessels containing the test item HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol) in inoculated mineral medium the mean concentration of dissolved organic carbon (DOC) varied between 24.7 and 29.0 mg/L over the exposure period of 28 days and thus was not significantly different from the initial mean DOC concentration of 28.6 mg/L measured on Day 0. Expressed as percentage DOC removal, mean values in the range from -1.4 to 14 % were noted. By the end of the test (Exposure Day 28), average biodegradation was 9 %.

Biodegradation in the Toxicity Control
In the toxicity control, containing both HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol) (corresponding to 50.8 % of total DOC) and the reference item sodium benzoate (corresponding to 49.2 % of total DOC) in inoculated mineral medium, the initial DOC concentration of 57.5 mg/L measured on Day 0 decreased to 27.9 mg/L on Day 14. Biodegradation amounted to 52 % within 14 days of exposure. By the end of the test (Exposure Day 28), average biodegradation was 55 %.

Thus, according to the test guidelines, the test item HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol) was not inhibitory to activated sludge at the tested concentration of 50 mg/L because degradation was >35 % within 14 days.

Measurement of pH and Dissolved Oxygen Concentration
All vessels started the test with a pH of 7.4. At the end of exposure (Day 28), pH values of 7.1 – 7.4 were measured.
The dissolved oxygen concentration was found to be in the range of 8.6 – 8.7 for all vessels at test start. At the end of exposure (Day 28), values of 8.4 – 8.5 mg O2/L were measured.

BOD5 / COD results

Results with reference substance:
In the procedure controls, average biodegradation of the reference item sodium benzoate was 92 and 98 % by Exposure Day 3 and 14, respectively, thus confirming suitability of the activated sludge (≥70 % degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 98 %.

Any other information on results incl. tables

Dissolved Organic Carbon (DOC) Concentration Measured in the Test Vessels

 

DOC[mg/L]1

 

Test Item

Procedure Control

Inoculum
Control

Toxicity
Control

Time

Replicate No.

Replicate No.

Replicate No.

Replicate No.

[Days]

1

2

Mean2

1

2

Mean2

1

2

Mean

12

 

 

 

 

 

 

 

 

 

 

 

0

29.1

30.0

28.6

30.5

29.9

29.3

0.6

1.2

0.9

57.5

3

29.1

29.7

28.8

3.4

2.8

2.5

0.6

0.5

0.6

--

5

28.7

29.3

28.3

--

--

--

0.7

0.7

0.7

--

7

27.9

28.4

26.8

2.6

1.1

0.5

1.8

1.0

1.4

26.9

10

28.5

28.7

27.5

--

--

--

1.5

0.8

1.2

--

12

25.4

26.1

24.7

--

--

--

1.1

1.0

1.1

--

14

28.4

28.7

27.8

1.7

1.0

0.6

0.8

0.7

0.7

27.9

18

29.4

29.8

29.0

--

--

--

0.5

0.7

0.6

--

21

26.6

27.4

26.3

--

--

--

0.7

0.7

0.7

--

28

26.7

27.2

26.2

1.8

1.0

0.6

0.8

0.8

0.8

26.2

1Mean values of at least duplicate measurements per sample

2Values corrected for the mean inoculum control

--Not determined

 

 Biodegradation of the Test Item

 

Percentage Biodegradation1

Time [Days]

Test Item

Reference Item

Toxicity Control

 

Replicate No.

Replicate No.

Replicate No.

 

1

2

Mean

1

2

Mean

1

0

0

0

0

0

0

0

0

3

-1

0

-1

91

92

92

--

5

0

2

1

--

--

--

--

7

6

7

6

96

101

98

53

10

3

5

4

--

--

--

--

12

13

14

14

--

--

--

--

14

2

4

3

97

99

98

52

18

-2

0

-1

--

--

--

--

21

8

8

8

--

--

--

--

28

8

9

9

97

99

98

54

1Corrected for the inoculum control

--Not determined

 

pH and Dissolved Oxygen Concentration of the Test Solutions at the Start and End of the Test

Replicate No.

Identification

pH

Dissolved Oxygen Concentration

Start

End

Start

End

1

Test item

7.4

7.1

8.7

8.4

2

Test item

7.4

7.1

8.7

8.4

1

Procedure control

7.4

7.4

8.7

8.4

2

Procedure control

7.4

7.4

8.7

8.4

1

Inoculum control

7.4

7.2

8.7

8.5

2

Inoculum control

7.4

7.2

8.6

8.5

1

Toxicity control

7.4

7.2

8.7

8.5

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was investigated for its ready biodegradability in a 28-Day DOC Die-Away Test according to the OECD Guideline for Testing of Chemicals, No. 301 A (1992), the Method C.4-A of Commission Regulation (EC) No 440/2008 and the US EPA OPPTS 835.3110 Ready Biodegradability, Paragraph (l) (1998). The test item was found to be not readily biodegradable after 28 days of exposure to activated sludge under the conditions of the conducted DOC Die-Away Test.
Executive summary:

The test item was investigated for its ready biodegradability in a 28‑Day DOC Die-Away Test according to the OECD Guideline for Testing of Chemicals, No. 301 A (1992), theMethod C.4-A of Commission Regulation (EC) No 440/2008 and the US EPA OPPTS 835.3110 Ready Biodegradability, Paragraph (l) (1998).

 

In the test vessels containing the test item in inoculated mineral mediumthe mean concentration of dissolved organic carbon (DOC) varied between 24.7 and 29.0 mg/L over the exposure period of 28 days and thus was not significantly different from the initial mean DOC concentration of 28.6 mg/L measured on Day 0. Expressed as percentage DOC removal, mean values in the range from -1.4 to 14  % were noted.By the end of the test (Exposure Day 28), average biodegradation was 9 %.

 

Consequently, the test item was not biodegradable under the test conditions within 28 days.

 In the toxicity control, containing both test item and the reference item sodium benzoate,the initial DOC decreased by 52 % within 14 days of exposure. Thus, the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 50 mg/L.

 

In the procedure controls, average biodegradation of the reference item sodium benzoate was 92 and 98 % by Exposure Day 3 and 14, respectively, thus confirming suitability of the activated sludge (≥70 % degradation by Exposure Day 14). By the end of the test (Exposure Day 28), average biodegradation was 98 %.