Reaction mass of dioctyl hydrogen phosphate and octyl dihydrogen phosphate

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:

2017-10-19-2017-02-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2016-06-29

Test material

Test material information

Constituent 1

Reference substance name:

Dioctyl hydrogen phosphate

EC Number:

221-485-7

EC Name:

Dioctyl hydrogen phosphate

Cas Number:

3115-39-7

Molecular formula:

C16H35O4P

IUPAC Name:

Dioctyl hydrogen phosphate

Constituent 2

Reference substance name:

Octyl dihydrogen phosphate

EC Number:

223-638-3

EC Name:

Octyl dihydrogen phosphate

Cas Number:

3991-73-9

Molecular formula:

C8H19O4P

IUPAC Name:

octyl dihydrogen phosphate

impurity 1

Reference substance name:

Trioctyl phosphate

EC Number:

217-305-1

EC Name:

Trioctyl phosphate

Cas Number:

1806-54-8

Molecular formula:

C24H51O4P

IUPAC Name:

trioctyl phosphate

impurity 2

Reference substance name:

Octanol

EC Number:

249-405-6

EC Name:

Octanol

Cas Number:

29063-28-3

Molecular formula:

C8H18O

IUPAC Name:

octan-1-ol

Test material form:

liquid

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge from the municipal wastewater treatment plant AZV Staufener Bucht; The treatment plant clarifies predominantly domestic wastewater and has a capacity of 140,000 inhabitant equivalents.
- Pretreatment: The activated sludge was washed twice with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.
- Concentration of sludge: 30 mg dry solids per litre

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

56.3 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Experimental set up
The CO2-free air production system consists of an air compressor, three 1000 mL gas wash bottles filled with dry soda lime in series followed by one bottle filled with 0.1 M NaOH (sodium hydroxide). At the end of the system is one gas wash bottle filled with demineralised water, followed by an empty one to catch any drops of condensation water. A color change of the soda lime from white to blue indicates that the CO2 absorption capacity is depleted. The CO2-free air is passed on to an air distributor with two input and 22 output channels and through PE-tubes Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septa were used as reactors. The liquid volume was fixed as 1500 mL each. Mixing was performed by magnetic stirrers with 2 cm stir bars.
The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series each filled with 200 mL 0.2 M NaOH. Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 mL PE syringes.

Procedure
In total two reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound, one reactor containing test item and reference compound (toxicity control) and one reactor without inoculum containing test item and a toxicant (abiotic control) were set up. 11.3 mL activated sludge was filled up to 1500 mL with 1488.7 mL mineral medium corresponding to 30 mg/L dry solids. Only the abiotic control vessel was filled with 1500 mL mineral medium without inoculum. The system was sealed and aerated with CO2-free air overnight. The reactors were kept mixed with magnetic stirrers. On the next day, the absorber wash bottles were filled with 0.2 M NaOH and the test substance was added into the two test vessels, into the toxicity control vessel and the abiotic control vessel. The reference compound was added into the reference vessels and into the toxicity control vessel and the toxicant into the abiotic control vessel. The aeration rate was kept at a rate of 30 - 100 mL / min (1.6 - 5.5 bubbles / second) and determined visually daily on working days. The determination by counting the gas bubbles over a defined period using a stop watch was made on days 6 and 26. The CO2-free air production system, the air-tightness of the whole experimental set-up, the aeration of the absorber flasks and the magnetic stirrers were controlled daily on working days. At the beginning of the study the IC concentration of the 0.2 M NaOH used for the CO2-absorption flasks was determined as 5.3 mg/L. The IC in the reactors at the beginning of the test was 0.865 mg/L. On the 4th, 7th, 11th, 14th, 21st and 28th day 4 mL NaOH from the first of two CO2-absorber flasks connected in line was sampled and the IC's were determined. The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air. On the 28th day 2 mL of 4M hydrochloric acid (HCl) was added into each reactor to release the CO2 dissolved in water. On day 29 the IC was determined in both CO2-absorber flasks in line.
IC measurement was performed with a total carbon analyser (TOC-5050A Shimadzu) by purging the inorganic carbon with H3PO4 (25%) using a non-dispersive infrared (NDIR) detector.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

83.8

Sampling time:

28 d

Details on results:

In order to find the exact position of the 10-d window the degradation values of day 1 to 3 and day 12 to 13 were calculated by interpolation. On day 2, the degradation was for the first time greater than 10%. Therefore, the end of the 10-d window is on day 12. On day 12, a mean degradation of 60.4% was calculated

Toxicity control
The degradation extent in the toxicity control was 79.5% within 14 days. According to the guideline the test substance had no inhibitory effect on the inoculum.

Abiotic control
The degradation extent in the abiotic control was 28.3% on day 28 after acidification

Reference item
The reference compound sodium benzoate reached the pass level for ready biodegradability within 4 days

Blank
The mean CO2-evolution of the blank flasks was 31.9 mg/L on day 28 after acidification

Criteria of validity
The IC content in the test vessel was less than 5% of the TOC introduced with the test item.
The CO2 evolution in the inoculum blank at the end of the test was below 40 mg/L.
The difference of extremes of replicate values of the test item at the end of the test was less than 20%.
The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 4.
The degradation extent in the toxicity control was above 25% in 14 days based on ThCO2
The test is valid according to OECD Test Guideline 301 B (July 1992).

Any other information on results incl. tables

Table 1: Biodegradation after x days (% of ThCO2)

Reactor	Day	0	4	7	11	14	21	28	29
19	Test flasks	0	25.3	49	59.1	62.6	76.4	83	85.4
20		0	22.4	50.4	59.7	62	85.6	81.4	82.1
4	Reference flasks	0	68.7	84.1	86.3	87.3	101.4	89.5	89.9
5		0	80.9	89.9	92.2	92.2	106.6	97.8	99.3
6		0	73.9	86.4	86.3	88.9	107.2	96.3	98.3
21	Toxicity control test item reference item	0	45	67.9	76.9	79.5	92.1	86.3	88.2
22	Abiotic control	0	6.3	6.4	7.2	8.2	11.3	13.4	28.3

Table 2: Interpolated degradation extents (% of ThCO2)

Reactor	Day	0	1	2	3	4	11	12	13	14
19	Test flasks	0	6.3	12.6	18.9	25.3	59.1	60.3	61.4	62.6
20		0	5.6	11.2	16.8	22.4	59.7	60.5	61.2	62
Mean degradation extent		0	6	11.9	17.9	23.8	59.4	60.4	61.3	62.3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The degradation of the test item at the end of the test was 83.8% (28 d after acidification, mean of two replicates).
The test item reached the criteria for ready biodegradability (60% of ThCO2 within a 10-d window).
The degradation of the toxicity control after 14 days was 79.5%. The test item had no inhibitory effect on the inoculum according to the criterion of the guideline.
The degradation of the abiotic control at the end of the test was 28.3%

Executive summary:

The ready biodegradability of the reaction mass of dioctyl hydrogen phosphate and octyl dihydrogen phosphate was tested in a CO2 evolution test according to OECD TG 301B. Activated sludge of a sewage treatment plant served as inoculum. Samples of the test substance and positive controls (sodium benzoate) were incubated for 28 days at 20.6 - 24 °C in diffuse light. Two replicates were set up. CO2 evolution was measured by IC measurements at days 0, 4, 7, 11, 14, 21, 28, 29.

After 28 days, the reaction mass of dioctyl hydrogen phosphate and octyl dihydrogen phosphate was degraded by 83.8%. 10% biodegradation was estimated to be reached on day 2 and 60% by day 12, the 10 day window being passed.

Based on the test results, the reaction mass of dioctyl hydrogen phosphate and octyl dihydrogen phosphate is readily biodegradable according to criteria of OECD test guidelines on biodegradation.

The study is assessed as accepable. It satisfies the requirements of OECD test guideline 301B for ready biodegradability without restrictions.