Trihexyl phosphate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

08 Aug - 19 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 17 Jul 1992

Deviations:

yes

Remarks:

Detailed information are summarized in section "Any other information on material and methods incl tables"

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

adopted 30. May 2008

Deviations:

yes

Remarks:

Detailed information are summarized in section "Any other information on material and methods incl tables"

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz, Germany

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf. Date of collection: 15. Sep. 2016, batch no: 20160915
- Preparation of inoculum for exposure: inoculum was taken from its source,washed, aerated and the dry matter was determined; test vessels were filled with medium and inoculum and aerated afterwards.
- Pretreatment: sludge was filtrated, washed with tap water (2x), then washed with and re-suspended in test medium. It was then aerated until use for 96 hours with purified, CO2-free, moistened air to purge the system of CO2
- Concentration of sludge: 4600 mg suspended solids/L
- Inoculum concentration: 25.0 mg/L dry matter/L

Duration of test (contact time):

29 d

Initial conc.:

20 mg/L

Based on:

other: organic carbon

Initial conc.:

32.4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: according to the guideline
- Test temperature: 19.8 – 21.5 °C (see deviation in section „Any other information on material and methods incl. tables“)
- Suspended solids concentration: dry matter was determined as 4600 mg suspended solids/L
- Continuous darkness: incubated in the dark

TEST SYSTEM
- Culturing apparatus: 2000 mL-SCHOTT-flasks were used as test vessels, 100 mL scrubber flasks as absorbent vessels.
- Number of culture flasks/concentration: 2 (per test item, blank, apparatus blank and reference) and 1 reactor (per toxicity control and abiotic control)
- Method used to create aerobic conditions: The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened 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.

- Measuring equipment: 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, respectively (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) every month. After every start, quality control samples were measured.

- Details of trap for CO2 and volatile organics if used: The emitted CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels. The initial IC value of the 0.25 M NaOH was separately determined in each flask.

SAMPLING
- Sampling frequency: On the 0, 2nd, 4th, 7th, 9th, 11th, 15th, 18th, 23trd, 28th and 29 day
- Sampling method: From each front scrubber flask, 10 samples were taken in order to determine the emitted CO2. The sample volume was 1 mL. The resulting change in the volume of the front flask was considered in the calculation of emitted CO2. 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.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 2 reactors
- Toxicity control: yes, 1 reactor
- Reference control: yes, 2 reactors
- Abiotic control: yes, 1 reactor

Reference substance:

aniline

Remarks:

Phenylamine, C6H5NH2, CAS-No. 62-53-3

Parameter:

% degradation (CO2 evolution)

Value:

41

Sampling time:

29 d

Results with reference substance:

Degradation of the positive control was 65% after 9 days.

Biological Results

Table 1: Degradation values in %

Day	Positive Control 1	Positive Control 2	Positive Control Mean	Test 1	Test 2	Test Mean	Abiotic Control	Toxicity Control
2	-0.2	-0.3	-0.3	-0.2	-0.3	-0.3	1.8	2.3
4	9.2	20.8	15.0	0.2	-0.8	-0.3	1.7	8.2
7	42.6	54.1	48.3	1.3	-1.7	-0.2	1.4	28.4
9	64.5	66.0	65.2	0.5	-3.2	-1.4	1.0	33.2
11	74.5	75.1	74.8	1.0	-3.3	-1.1	0.9	40.3
15	83.7	81.6	82.6	3.3	-2.5	0.4	0.9	44.6
18	84.0	78.3	81.1	10.0	2.5	6.2	0.9	49.2
23	88.7	80.8	84.8	23.4	23.4	23.4	0.8	58.1
29	89.1	81.5	85.3	38.2	42.9	40.5	1.6	68.6

 

Table 2: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	4.7%	Yes
Percentage degradation of the reference compound has reached the pass levels by day 14.	9 days	Yes
The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC, and the total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.	4.37% IC content of test item solution in medium   9.5 mg/L CO2 emitted by the controls	Yes

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Description of key information

Not readily biodegradable (Pass level OECD301B test reached after 38 d with 61% (enhanced ready biodegradable)). Since the pass level of the OECD 301B test reached after a prolonged test duration up to 38 days, it can be considered that the substance will fully mineralize under environmental conditions within a short time period and thus persistency in the environment can be excluded.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater

Additional information

Two GLP guideline studies according to OECD 301 B are available investigating the ready biodegradability of the substance (CAS 2528-39-4). In both studies the pass level was not reached within 28 days therefore the OECD criteria for being readily biodegradable were not fulfilled. 

The key study was performed as an enhanced study (LAUS GmbH, 2017a) inoculating a mixed microbial population of the activated sludge of a local municipal STP with a nominal concentration of 32.8 and 33.1 mg/L test substance and incubated for 38 d under controlled conditions in the dark. Degradation was followed by the determination of carbon dioxide (CO2) evolution after 0, 2, 6, 8, 10, 14, 17, 23, 29, 34, 37 and 38 d. An inoculum blank, a reference control and a toxicity control were run in parallel. The derived degradation rate was 54.9% after 29 d and 61.0% after 38 d. Since the test substance did not reach the pass level of 60% biodegradation within 28 days it is not considered to be readily biodegradable according to the OECD criteria. However, the degradation rate of 61% was reached after a test prolongation up to 38 d. Since the pass level reached after 38 days, it can be considered that the substance will fully mineralize under environmental conditions within a short time period and thus persistency in the environment can be excluded.

The supporting study confirms the results of the key study. This GLP guideline study is investigating the ready biodegradability of the substance according to OECD 301 B (LAUS GmbH, 2017b). A mixed microbial population of the activated sludge of a local municipal STP was inoculated with a nominal concentration of 32.4 mg/L test substance and incubated for 29 d under controlled conditions in the dark. Degradation was followed by the determination of carbon dioxide (CO2) evolution after 2, 4, 7, 9, 11, 15, 18, 23 and 29 d. An inoculum blank, a reference control and a toxicity control were run in parallel. The derived 29 d degradation rate was 41% for the test substance and 44% for the toxicity control after 14 d.

It can be concluded that the substance was not readily biodegradable under the chosen test conditions. Nevertheless, persistency under environmental conditions can be excluded due to the fact that the pass level was reached in one of the ready tests after 38 d with 61%.