Trihexyl phosphate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 - 06 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

yes

Remarks:

see section "Any other information on material and methods incl. tables".

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

2015

Deviations:

yes

Remarks:

see 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

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: test item was added to the test vessels directly
- Controls: yes,medium and inoculum

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: sewage treatment plant ESN (Stadtentsorgung Neustadt) in NW-Lachen-Speyerdorf, Germany; Date of collection: 05 Sep 2016
- Preparation of inoculum for exposure: inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter, the sludge was fed with synthetic seawage feed (also named as nutrient solution) 50 mL/L sludge. On the day of the experiment, the dry matter was determined once more.
- Initial biomass concentration: 2.66 g suspended solids/L (dry matter of sludge) and 1.33 g suspended solids/L (dry matter in the test)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.2 - 21.2 °C (main test)

pH:

7.8 - 7.9 (control, main test)
7.9 - 8.0 (positive control, main test)
7.5 - 7.7 (test item, main test)

Nominal and measured concentrations:

control, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel
- Material, size, headspace, fill volume: glass beakers were used as test vessels with a volume of 250 mL narrow-neck glass bottles as measuring flasks
- Aeration: with purified air, using Pasteur pipettes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): 2.66 g suspended solids/L for dry matter of sludge and 1.33 g suspended solids/L for dry matter in the test
- Nutrients provided for bacteria: nutrient solution according to the guidelinne OECD 209

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Other: for parameter see Table 1 in section "Any other information on material and methods incl tables"

EFFECT PARAMETERS MEASURED: oxygen consumption as a proxy for respiration rate aftere aeration time of 3 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study:
- Test concentrations: 1, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study:
At the beginning of oxygen measurement, the oxygen concentration in the test solutions was much lower than in the positive control and the control solutions. Therefore, duration of oxygen measurement in the test solutions was partly very short and the inhibition values in the test solutions were in a similar range at all concentrations and strongly scattering. The low oxygen concentration in the test solution was caused by the test items properties, a poorly soluble liquid. On the surface of the test solutions, a thin layer of test item on the surface of the test solution was present and the gas exchange between liquid phase and atmosphere was biased. Therefore, the oxygen uptake of the test solutions was reduced.

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Blank controls oxygen uptake rate: 28.87 mg oxygen/g sludge per h
- Coefficient of variation of oxygen uptake rate in control replicates: 2.5%
- Other: Biological results are summarized within table 1 insection "Any other information on results incl. tables"

Results with reference substance (positive control):

- Results with reference substance valid? The EC50 of the reference compound 3,5-Dichlorophenol should be in the range of 2 - 25 mg/L for total respiration. In the present test the EC50 of the reference compound was 11 mg/L; thus the test is valid.

Table 1: O2-Consumption, Inhibition second experiment (main test)

Vessel No.	Content	Concentration in mg/L nominal	O2 consumption in mg/(L*min)	O2 consumption in mg/(L*h)	Inhibition in %	pH
1	Blank Control	0	0.6336	38.015	0	7.8
2	Blank Control	0	0.6391	38.344	0	7.9
3	Positive Control	5	0.4959	29.752	22.5	7.9
4	Positive Control	10	0.3122	18.733	51.2	7.9
5	Positive Control	20	0.1679	10.072	73.8	8.0
6	Positive Control	40	0.1012	6.074	84.2	8.0
7	Blank Control	0	0.6328	37.969	0	7.9
8	Blank Control	0	0.6242	37.454	0	7.8
9	Test Item	1000	0.5961	35.764	6.9	7.7
10	Test Item	1000	0.6128	36.770	4.2	7.6
11	Test Item	1000	0.6060	36.360	5.3	7.6
12	Test Item	1000	0.6104	36.624	4.6	7.6
13	Test Item	1000	0.6065	36.390	5.2	7.6
14	Test Item	320	0.6234	37.401	2.6	7.6
15	Test Item	320	0.6068	36.406	5.2	7.6
16	Test Item	320**	0.6007	36.043	6.1	7.5
17	Test Item	320**	0.6166	36.998	3.6	7.6
18	Test Item	320**	0.6334	38.002	1.0	7.6
19	Test Item	100**	0.6216	37.296	2.9	7.6
20	Test Item	100**	0.6585	39.511	-2.9	7.7
21	Test Item	100	0.6104	36.626	4.6	7.6
22	Test Item	100	0.6322	37.929	1.2	7.6
23	Test Item	100	0.6327	37.961	1.1	7.6
24	Test Item	32	0.6249	37.497	2.3	7.6
25	Test Item	32	0.6189	37.136	3.3	7.6
26	Test Item	32	0.6167	37.005	3.6	7.6
27	Test Item	32	0.6175	37.051	3.5	7.6
28	Test Item	32	0.6289	37.735	1.7	7.6
29	Test Item	10	0.6292	37.749	1.7	7.6
30	Test Item	10	0.6488	38.925	-1.4	7.6
31	Test Item	10	0.6422	38.529	-0.3	7.6
32	Test Item	10	0.6456	38.734	-0.9	7.6
33	Test Item	10	0.6530	39.182	-2.0	7.6
34	Blank Control	0	0.6391	38.345	0	7.8
35	Blank Control	0	0.6710	40.261	0	7.8

** At the beginning of oxygen measurement, the oxygen concentration in the test solutions was much lower than in the positive control and the control solutions. Therefore, duration of oxygen measurement in the test solutions was partly very short

Table 2: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.	28 mg oxygen per one gram of activated sludge in an hour.	Yes
The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.	2.5%	Yes

Validity criteria fulfilled:

yes

Description of key information

EC50 (3 h): > 1000 mg/L (activated sludge, OECD 209)

NOEC (3 h): ≥ 1000 mg/L (activated sludge, OECD 209)

Additional information

One experimental study was conducted in accordance with OECD Guideline 209 under GLP conditions (LAUS GmbH, 2017f). The activated sludge was exposed to the test item at a concentration range of 10, 32, 100, 320, and 1000 mg/L representing the total microbial community. The respiration rate of each mixture was determined after aeration periods of 3 hours. The test item showed no statistically significant effects up to and including 1000 mg/L. As no inhibition was observed the EC50 and EC10 (3 h) is higher than 1000 mg/L. The NOEC (3 h) is equal or higher than 1000 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.