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Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Cas Number:

2409816-82-4

Molecular formula:

C26H42O4P2

IUPAC Name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Test material form:

liquid

Specific details on test material used for the study:

Name tributyl(ethyl)phosphonium diphenyl phosphate
Batch no. JXXX2021-015
CAS no. 2409816-82-4
Composition tributyl(ethyl)phosphonium diphenyl phosphate 98 %
Storage room temperature (20 ± 5 °C)
Expiry date 15. Mar. 2022
Stability stable under storage conditions
Appearance Light yellow liquid
Purity 98 %
Homogeneity homogeneous
Production date 15. Mar. 2021
EC no. 870-540-7
Molecular formula C26H42O4P2
Molecular weight 480 g/mol
Vapour pressure not stated
Solubility in solvents H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown; methanol: not stated
Stability in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated; methanol: not stated

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Specification:
Activated sludge from a biologic sewage treatment plant was used as inoculum. The chosen plant treats mostly domestic sewage.

Source:
The sludge was taken from the activation basin of the sewage treatment plant, sewage treat-ment plant, In den Seewiesen, 67482 Edenkoben.
Date of collection: 07. Jan. 2022, batch no: E20220107.

Pre-Treatment：
The sludge was filtrated through a cloth, washed with test medium (2x) and resuspended in test medium. It was then aerated until use. The dry matter was determined to contain 4.02 g of suspended solids/L.

Duration of test (contact time):

28 d

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

7.1 Preparations
The medium was prepared from the stock solutions. The stock solution of the positive control was prepared and its organic carbon was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined.
The test vessels were filled with medium and inoculum. Then, all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.

7.2 Experimental Parameters
Flask volume 1500 mL
Apparatus blanks 2, containing mineral medium only
Blank Controls 2, containing mineral medium and inoculum
Positive control flasks 2, containing positive control, mineral medium and inoculum
Test flasks 2, containing test item, mineral medium and inoculum
Abiotic control 1, containing test item, mineral medium and HgCl2
Toxicity control 1, containing test item, positive control, mineral medium and in-oculum
Inoculum concentration: 25.0 mg/L
Temperature 18.9 – 21.6 °C without direct lighting
Duration 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L of the test item.

7.3 Apparatus
The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, mois-tened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. To control the absence of CO2, the air was then led through a flask containing a solution of Ba(OH)2 before reaching the test vessels.
Magnetic stirrers were used to prevent deposition of inoculum.
The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels.

7.4 Sampling
From each front scrubber flask, 9 samples were taken in order to determine the emitted CO2 (on day 2, 4, 7, 9, 11, 14, 18, 23 and 29). The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2 (see also chapter 8.3.1).
On day 0, only one sample of 0,25 M NaOH was sampled. This measured value was used as start value for all treatments.
On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.

7.5 CO2 Determination
Analyses of the emitted CO2 were made by IC measurement using the carbon analyser TOC multi N/C 2100S, Analytik Jena. Each sample was measured in duplicate or triplicate, re-spectively (depending on the variation between the measured values). The carbon analyser was calibrated with freshly prepared reference solutions containing potassium hydrogen phthalate (TC), sodium hydrogen carbonate and sodium carbonate (IC). Quality control sam-ples were measured on each measuring day.

Results and discussion

Test performance:

PERFORMANCE OF THE STUDY
7.1 Preparations
The medium was prepared from the stock solutions. The stock solution of the positive con-trol was prepared and its organic carbon was measured. The inoculum was taken from its source, washed, aerated and the dry matter was determined.
The test vessels were filled with medium and inoculum. Then, all flasks were aerated for 72 hours with purified, CO2-free, moistened air to purge the system of CO2.
7.2 Experimental Parameters
Flask volume: 1500 mL
Apparatus blanks: 2, containing mineral medium only
Blank Controls: 2, containing mineral medium and inoculum
Positive control flasks: 2, containing positive control, mineral medium and inoculum
Test flasks: 2, containing test item, mineral medium and inoculum
Abiotic control: 1, containing test item, mineral medium and HgCl2
Toxicity control: 1, containing test item, positive control, mineral medium and inoculum
Inoculum concentration: 25.0 mg/L
Temperature : 8.9 – 21.6 °C without direct lighting
Duration : 28 days
The test was performed with a nominal start concentration of 20 mg organic carbon/L of the test item.

The following amounts of test item and positive control were added to the flasks:
Table 7.2 a Amounts of test item and positive control in the flasks
Flask Positive Control 1 Positive Control 2 Test 1 Test 2 Abiotic Control Toxicity Control
Amount Test item
in mg / L -- -- 30.9 31.0 30.9 31.3
Amount Aniline
in mg / L 25.2 25.2 -- -- -- 25.2
organic C (calculat-ed) in mg / L 20.0 20.0 20.1 20.1 20.1 40.3
The added amount of test item and positive control is based on the TOC content (total organic car-bon. The TOC content of the poorly soluble test item was calculated by molecular formula. The TOC content of the water-soluble positive control was determined by TOC measurement of a stock solu-tion.
Note: All calculations are performed with unrounded values. Therefore, recalculation with rounded values may lead to slightly different results.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

32

Sampling time:

28 d

Details on results:

Results for the Test Item tributyl(ethyl)phosphonium diphenyl phosphate
 The test item tributyl(ethyl)phosphonium diphenyl phosphate is considered as “not readily biodegradable“.
 The degree of biodegradation reached 32 % after 28 days.
 The 10-day-window began on day 3, at its end, 30 % degradation were reached, miss-ing the pass level of 60 % given in the OECD guideline.
 Degradation missed 60 % within 28 days, too. Therefore, the test item is considered as “not ultimately biodegradable, within 28 days”, as well.
 Abiotic degradation was not observed.

Validity:
All validity parameters and values are presented in the following table:
Table Validity
Parameter Criterion Found Assessment
IC content of test item solution in medium < 5% of TC 0.21 % valid
CO2 emitted by the controls < 70 mg/L 17.8 mg/L valid
Difference within replicates < 20% 4.2 % valid
Degradation of positive control ≥ 60% ≤ 14 days 13 days valid
Degradation in the toxicity flask on day 14 > 25% 46.3 % non - toxic

Any other information on results incl. tables

Calculation Results

1.1.1     Emitted Carbon in mg/L

In the following table, the calculated emitted carbon (from net IC given in chapter 8.1.2 and equation stated in chapter 8.2.1) is presented.

Table 8.3‑a    Emitted carbon in mg/L

Day	Blank Control 1	Blank Control 2	Positive Control 1	Positive Control 2	Test 1	Test 2	Abiotic Control	Toxicity Control
2	0.57	0.38	0.41	-0.04	0.32	0.71	0.21	0.02
4	0.64	0.61	1.63	1.16	2.31	4.98	0.11	1.26
7	1.30	0.90	10.63	6.12	5.65	6.39	0.16	9.80
9	2.01	1.45	12.47	7.76	6.83	7.46	0.10	15.03
11	2.06	2.04	15.24	9.74	8.18	7.77	0.18	18.74
14	2.78	2.98	17.22	14.06	9.16	8.95	0.21	21.54
18	3.86	3.22	18.67	14.14	9.40	9.83	-0.17	23.18
23	4.89	3.80	19.83	17.16	10.24	11.21	0.01	25.30
29	5.00	4.73	19.37	17.02	10.95	11.81	-0.59	26.44

 

8.3.2 Degradation Values
In the following table, the percentage biodegradation is presented:
Table 8.3 b Degradation values in %

Day	Positive Control 1	Positive Control 2	Positive Control Mean	Test 1	Test 2	Test Mean	Abiotic Control	Toxicity Control
2	-0.3	-2.6	-1.4	-0.8	1.2	0.2	1.1	-1.1
4	5.0	2.7	3.9	8.4	21.6	15.0	0.5	1.6
7	47.7	25.1	36.4	22.6	26.3	24.4	0.8	21.6
9	53.8	30.2	42.0	25.4	28.5	26.9	0.5	33.0
11	66.1	38.5	52.3	30.5	28.4	29.5	0.9	41.4
14	71.8	56.0	63.9	31.2	30.1	30.7	1.1	46.3
18	75.7	53.1	64.4	29.1	31.2	30.2	-0.9	48.7
23	77.5	64.1	70.8	29.3	34.1	31.7	0.1	52.0
29	72.7	60.9	66.8	30.3	34.5	32.4	-2.9	53.5

Because the values of day 29 are the sum of the IC values in both scrubber flasks, an in-crease (IC values in flasks B of the test higher than in those of the control) or a decrease (IC values in flasks B of the test lower than in those of the control) of degradation can be observed.
As the measured IC values in the abiotic control are very low, measurement uncertainties lead to negative degradation values while in fact no degradation has taken place.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met.

Interpretation of results:

not readily biodegradable

Conclusions:

All validity criteria were met.
Degradation behavior of positive control and toxicity control was normal. Abiotic degrada-tion was not observed. Both replicates of the test item showed very good correspondence.
If degradation in the toxicity flask is below 25 % after 14 days, the test item can be consid-ered as toxic towards the inoculum. As degradation in the toxicity flask was 46.3 % after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 31.3 mg/L”.
Ready biodegradability is defined in the guidelines as degradation surpassing 60% within 10 days after reaching a level of 10 %. The 10-day-window began on day 3, at its end, 30 % degradation were reached, missing the pass level of 60 % given in the OECD guideline. Therefore, the test item tributyl(ethyl)phosphonium diphenyl phosphate can be considered as “not readily biodegradable”. Degradation missed the pass level of 60 % within 28 days, though. Therefore, the test item can be considered as “not ultimately biodegradable, within 28 days, as well.
No observations were made which might cause doubts concerning the validity of the study outcome.

The result of the test can be considered valid.

Executive summary:

The test item tributyl(ethyl)phosphonium diphenyl phosphate was tested using a concentra-tion of nominally 20 mg organic carbon/L tributyl(ethyl)phosphonium diphenyl phosphate in test medium following OECD 301B and EU-Method C.4-C.

Aniline was chosen as positive control.

Activated sludge was used as inoculum (concentration in the test 25.0 mg dry matter/L). The test was left running for 28 days.
All validity criteria were met. Degradation of the positive control surpassed the pass level of 60 % after 13 days.

The following data were determined for the test item tributyl(ethyl)phosphonium diphenyl phosphate:
10-day-window:                                                                                       day 3 – 13

degradation at the end of 10-day-window                                                         30 %

degradation at the end of the test                                                                    32 %

pass level following guideline:   60 % at the end of 10-day-window for pure substances
                                                    respective 60 % at the end of the test for mixtures

Therefore, when applying the 10-day-window, the test item tributyl(ethyl)phosphonium diphenyl phosphate is not readily biodegradable following OECD 301B and EU C.4-C re-spectively. As degradation missed the pass level of 60% in the course of the test, tributyl(ethyl)phosphonium diphenyl phosphate is considered as not ultimately biode-gradable, within 28 days.