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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:
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:
2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) and bisphenol A concentrations were analytically monitored on several occasions during the test.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
None
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
The inoculum was obtained on 10 November 2015 from the activated sludge of the biological wastewater treatment plant from Mourenx (France, 64) which handles predominantly domestic sewage.

On arrival at the laboratory, the sludge (around 1 L) was centrifuged at approximately 20°C for 10 min at 1000 g. The pellet was re-suspended in mineral medium in order to keep the concentration unchanged with respect to the sample. The inoculum was preconditioned for 48 hours (aeration) prior to the start of the experiment.

Sub-samples of the homogenised sludge were dried in an oven at approximately 105°C and the suspended solids content was determined to be 4.76 g/L prior to use.

The inoculum was not adapted to the test item before the biodegradation test.
Duration of test (contact time):
28 d
Initial conc.:
110 mg/L
Based on:
other: Analytically determined
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: as described in the OECD 301 guideline
- Additional substrate: none
- Solubilising agent (type and concentration if used): none
- Test temperature: 22.2°C - 22.9°C (min - max)
- pH: 7.3 - 7.8 (min - max)
- pH adjusted: yes/no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes/no

TEST SYSTEM
- Culturing apparatus: Respirometer BSBdigi Selutec® where the temperature was monitored over the entire duration of the test using a digital maximum/minimum calibrated thermometer
- Number of test item concentrations: 1 (110 mg/L)
- Number of culture flasks/concentration (medium + inoculum + test item): 2
- Number of abiotic flasks (medium + test item): 1
- Number of respiratory blanks (medium + inoculum): 2
- Number of toxicity controls (medium + inoculum + sodium benzoate + test item): 2
- Number of reference controls (medium + inoculum + sodium benzoate): 2

SAMPLING
- Sampling frequency: D0 and D28 for analytical determination of 2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) and bisphenol A
Reference substance:
benzoic acid, sodium salt
Preliminary study:
No preliminary study was carried out.
Test performance:
All validity criteria were met.
Key result
Parameter:
% degradation (O2 consumption)
Value:
5
Sampling time:
28 d
Key result
Parameter:
% degradation (test mat. analysis)
Value:
50
Sampling time:
28 d
Details on results:
CHNS analysis indicated that C, H and O represented 71.7%, 7.7% and 20.6% of the test item (w/w), i.e. an approximative theoretical formula: C18H23O4. ThOD of the test item was 2.32 mg O2 / mg test item. Concentration of 2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) was 110 mg/L at D0 (i.e. near the water solubility limit, as expected) and around 55 mg/L at D28, suggesting degradation of the substance. Traces of bisphenol A were found at D0 and no bisphenol A remained at D28. Ultimate biodegradation was only 5%. The test item is considered not readily biodegradable.
Results with reference substance:
Biodegradation of the reference substance was 95.3% after 28 days incubation.

Table 1: Results of CHNS analysis. Oxygen was back calculated as follows: O = 100 - N - C - H - S.

Component

Component content (% w/w)

Mean

SD

RSD (%)

Measure 1

Measure 2

Measure 3

N

<DL

<DL

<DL

NA

NA

NA

C

72.1

71.3

71.8

71.73

0.404

0.56

H

8.1

7.3

7.7

7.70

0.400

5.19

S

<DL[WN1] 

<DL

<DL

NA

NA

NA

< DL Nitrogen (0.13%): concentration lower than the Detection Limit of the analytical method

< DL Sulfur (0.79%L): concentration lower than the Detection Limit of the analytical method

NA: Not Applicable

Table 2: Results of NPOC analysis 

Test solution

NPOC concentrations (mg/L)

Control

<LD

Test item

84.2

< DL NPOC (0.44 mg/kg): concentration lower than the Detection Limit of the analytical method

Table 3: Concentrations of 2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) at D0 and D28 in the inoculated bioreactors

Sample time

(days)

Test solution

Test item concentrations

T0

Ft

110 mg/L

T28

Ft1

53.0 mg/L

Ft2

58.6 mg/L 

Table 4: Concentrations of bisphenol A at D0 in the inoculated bioreactors 

Sample time

BPA concentration(µg/L)

Dilution

Mean

SD

RSD (%)

Measure 1

Measure 2

Measure 3

T0

7.0947

7.1817

6.9697

2

14.2

0.21

1.5

T0 + 1h30

9.6354

8.5814

8.1354

2

17.6

1.5

8.8

T0 + 5h30

44.9987

44.5427

44.8606

2

89.6

0.47

0.52

T0 + 6h30

42.6607

43.4182

43.0045

2

86.1

0.76

0388

< DL (0.68 µg/L): concentration lower than the Detection Limit of the analytical method

< QL (2.25 µg/L): concentration lower than the Quantification Limit of the analytical method

 

 

Table 5: Concentrations of bisphenol A at D28 in the inoculated bioreactor

Test solution

BPA concentration(µg/L)

Dilution

Mean

SD

RSD (%)

Measure 1

Measure 2

Measure 3

Ft1

<DL

<DL

<DL

2

NA

NA

NA

Ft2

<DL

<DL

<DL

2

NA

NA

NA

Fb1

<DL

<DL

<DL

2

NA

NA

NA

Fb2

<DL

<DL

<DL

2

NA

NA

NA

< DL (1.18 µg/L): concentration lower than the Detection Limit of the analytical method

< QL (3.93 µg/L): concentration lower than the Quantification Limit of the analytical method

NA: Not Applicable

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) is not considered readily biodegradable. Half of the initial compound quantity initially introduced in the reactors disappeared after 28 days. Only traces of bisphenol A were found at the beginning of the test. Bisphenol A was not found in the reactors at the end of the test.
Executive summary:

The study was conducted in accordance with procedures outlined in OECD Guideline 301F (1992). The ready biodegradability of 2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) was assessed over a 28 days period. As the test item is a clear solid at 20°C, it cannot be directly added to each test bioreactor. The test item preparation was thus adapted from the "flask method with slow stirring conditions" described in the OECD 123 guideline. As a consequence, additional analytical works were performed in order to confirm the test item concentrations at Day 0 and Day 28 and to confirm ThOD of the substance. In addition, analysis was performed to check Bisphenol A formation over the test period. Indeed, Bisphenol A was hypothesized to be a metabolite of the test item in reason of its structural similarity.

A total of 7 bioreactors were used: 2 each for test item, blank control and reference item control and one for toxicity control. Test item was introduced close to its water solubility limit.

Analysis of the2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) initial concentrations (110 mg/L) confirmed that it was introduced close to its saturation in water. Test item biodegradation after 28 days was 5%. Biodegradation of the reference item was 97.2%. Biodegradation of the toxicity control was 42.8%. The test item was not readily biodegradable under the conditions of the test as it failed to achieve a transition from 10% to 60% degradation in a 10-day window during the 28 days. The test item was not considered to be inhibitory to the microbial inoculum since the biodegradation observed in the toxicity control bioreactor was not less than 39.6% occurred within 14 days.

Analysis of the2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) concentration after 28 days incubation with the inoculum showed that around 50% of the initial quantity introduced in the bioreactors had disappeared. Analysis showed a total absence of Bisphenol A at the end of the test.

The definitive test met the validity criteria of the test guideline detailed as follows:

- the difference of extremes of replicate values of the removal of the test item at the end of the test was less than 20%;

- the percentage degradation of the reference compound has reached the pass level by day 14 with 92.3%;

- the biodegradation observed in the toxicity control bioreactor was=25% by day 14 as it was 39.6%;

- the oxygen uptake of the inoculum blank was not greater than 60 mg/L in 28 days as it was 7.6 mg/L;

- the pH values were not outside the range 6-8.5 with min 7.3 and max 7.8

Description of key information

The study was conducted in accordance with procedures outlined in OECD Guideline 301F (1992). The ready biodegradability of 2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) was assessed over a 28 days period. As the test item is a clear solid at 20°C, it cannot be directly added to each test bioreactor. The test item preparation was thus adapted from the "flask method with slow stirring conditions" described in the OECD 123 guideline. As a consequence, additional analytical works were performed in order to confirm the test item concentrations at Day 0 and Day 28 and to confirm ThOD of the substance. In addition, analysis was performed to check Bisphenol A formation over the test period. Indeed, Bisphenol A was hypothesized to be a metabolite of the test item in reason of its structural similarity.

A total of 7 bioreactors were used: 2 each for test item, blank control and reference item control and one for toxicity control. Test item was introduced close to its water solubility limit.

Analysis of the2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) initial concentrations (110 mg/L) confirmed that it was introduced close to its saturation in water. Test item biodegradation after 28 days was 5%. Biodegradation of the reference item was 97.2%. Biodegradation of the toxicity control was 42.8%. The test item was not readily biodegradable under the conditions of the test as it failed to achieve a transition from 10% to 60% degradation in a 10-day window during the 28 days. The test item was not considered to be inhibitory to the microbial inoculum since the biodegradation observed in the toxicity control bioreactor was not less than 39.6% occurred within 14 days.

Analysis of the2,2'-Isopropylidenebis(p-phenyleneoxy)diethanol) concentration after 28 days incubation with the inoculum showed that around 50% of the initial quantity introduced in the bioreactors had disappeared. Analysis showed a total absence of Bisphenol A at the end of the test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information