Trihexyl phosphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Feb – 03 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 2006, corrected: 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations and the control
- Sampling method: For method A: 100 mL of algea solution were sampled and for method B: 50 mL.
- Others: See Section "Any other information on material and methods incl tables"

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Saturated solution was performed: 106 µL/L (corresponding to the load of 100 mg/L and based on a density of 0.94 g/mL) on the surface of algal medium and stirred slowely for 23 h. The lower phase was used as test solution. The lower treatments were prepared by dilution of this saturated solution with algal medium; 700 mL test item solution was mixed with appropriatre amount of algae pre-culture (1.81 mL).
- Controls: yes, medium

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Desmodesmus subspicatus SAG 86.61
- Source: MBM Sciencebridge GmbH, Institut für Pflanzenphysiologie of Universität Göttingen, Germany
- Age of inoculum (at test initiation): 4 day (preculture)
- Method of cultivation: Four days before the start of each test, an liquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.3 – 22.0 °C

pH:

7.3 - 8.0 (control)
7.6 - 8.7 (test concentrations)

Nominal and measured concentrations:

control, 1.0, 3.2, 10, 32 and 100 mg/L (nominal)
control, n.d., n.d., 23.0, 82.6 and 179.9 µg/L (geometric mean)
n.d.= not determinable due to contamination

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with perforated plastic foil)
- Material, size, headspace, fill volume: glass flasks total volume 65 mL, test volume: 45 mL, headspace 20 mL
- Initial cells density: 4.5x 10E+03
- Control end cells density: 39.1724 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Culture medium different from test medium: no culture and test medium were the same

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continously
- Light intensity and quality: 4500 lux

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: no range-finding test was performed

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

160 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

lower phase of saturated test solution was tested

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

95 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

lower phase of saturated test solution was tested

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

82.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

lower phase of saturated test solution was tested

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

110 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

lower phase of saturated test solution was tested

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

69 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

lower phase of saturated test solution was tested

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

82.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

lower phase of saturated test solution was tested

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no, all algae cells appeared normal and healthy

Results with reference substance (positive control):

The EC50 value was 0.80 mg/L for growth rate and 0.54 mg/L for yield and therefore within the recommended range (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software. Calculation was performed with Microsoft Excel® and ToxRat® Professional, version 3.2.1.

Analytical Results:

The measured concentrations in the replicates including algae were below the expected range determined during validation of the method. This was probably caused by adsorption of test item onto the algal cells. For elimination of the algae cells, the samples were centrifuged before measurement. The measured concentrations in the replicates without algae were within the expected range determined during validation of the method.

During the measurement after 24 and 48 h the sensitivity was low. Due to the time consuming extraction before measurement, no new measurement of the samples was possible. Therefore, for the measured values after 72 h, a fresh calibration was performed. The values after 24 and 48 h were not used for evaluation of the test item concentrations during the study.

At the end of the test the measured concentrations in the treatments 1.0 mg/L and 3.2 mg/L nominal concentrations without algae were biased caused by contamination. Therefore, evaluation was only performed using the three highest treatments. Determination of the results was based on the geometric mean of the measured concentrations after 0 and 72 h in the three highest treatments without algae.

Table 1: Measured Test item Concentrations with algae

Nominal Concentration Test Item	Measured Concentration Test Item t = 0 h	Measured Concentration Test Item t = 24 h	Measured Concentration Test Item t = 48 h	Measured Concentration Test Item t = 72 h
mg/L	μg/L	μg/L	μg/L	μg/L
Blank control	<LOQ	<LOQ*	<LOQ*	8.52**
1.0	<LOQ	<LOQ*	<LOQ*	8.70**
3.2	2.64	<LOQ*	<LOQ*	<LOQ
10	10.08	8.87*	4.16*	4.21
32	24.96	16.76*	19.16*	15.03
100	87.89	75.27*	80.78*	63.30

LOQ (Limit of Quantification) = 1.9 μg/L

* Due to the low sensitivity, the values after 24 and 48 hours were not used for evaluation of the test item concentrations during the study.

** Due to a contamination, an unexpected high value was determined at the end of the experiment. Therefore, these values were excluded from any further calculation.

Table 2: Measured Test item concentrations without algae

Nominal Concentration Test Item	Measured Concentration Test Item t = 0 h	Measured Concentration Test Item t = 24 h	Measured Concentration Test Item t = 48 h	Measured Concentration Test Item t = 72 h
mg/L	μg/L	μg/L	μg/L	μg/L
1.0	3.23	4.00*	2.06*	14.34**
3.2	8.98	7.51*	5.06*	11.02**
10	27.37	17.16*	12.56*	19.30
32	85.03	59.16*	69.97*	80.15
100	248.30	224.13*	217.95*	130.35

* Due to the low sensitivity, the values after 24 and 48 hours were not used for evaluation of the test item concentrations during the study.

** Due to a contamination, an unexpected high value was determined at the end of the experiment. Therefore, these values were excluded from any further calculation.

Table 3: Geometric mean of measured concentrations without algae of 0 and 72 hours

Nominal Concentration Test Item	Geometric Mean 0 h and 72 h
mg/L	μg/L
Blank control	--
1.0	--
3.2	--
10	23.0
32	82.6
100	179.9

 

Biological Results:

Table 4 Cell number/mL

Nominal Concentration	Parameter	Cell Number/mL
mg/L		0 h	24 h	48 h	72 h
Blank control*	Mean	4460	11512	78888	391724
Blank control*	SD	0	790	12771	79335
1.0*	Mean	4460	11730	87670	339280
1.0*	SD	0	467	5275	66383
3.2*	Mean	4460	12050	95810	366660
3.2*	SD	0	354	3041	90510
10	Mean	4460	11360	94333	420193
10	SD	0	819	4156	36595
32	Mean	4460	11107	77520	314213
32	SD	0	1867	8678	53301
100	Mean	4460	10260	17593	29113
100	SD	0	716	2320	7581

SD = Standard Deviation

* Due to low growth of one replicate it was excluded from further evaluation.

Table 5: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	88	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	34%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	5%	yes

Validity criteria fulfilled:

yes

Description of key information

ErC50 (72 h) = 160 µg/L (Desmodesmus subspicatus, OECD 201, geometric mean)

ErC10 (72 h) = 95 µg/L (Desmodesmus subspicatus, OECD 201, geometric mean)

Key value for chemical safety assessment

EC50 for freshwater algae:

160 µg/L

EC10 or NOEC for freshwater algae:

95 µg/L

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 (GLP) using the freshwater algae Desmodesmus subspicatus (LAUS GmbH, 2017e). The definitive test was carried out with the following test item concentrations: 1.0, 3.2, 10, 32, and 100 mg/L including a control. The measured concentrations in the replicates without algae were within the expected range determined during validation of the method. At the end of the test the measured concentrations in the treatments 1.0 mg/L and 3.2 mg/L nominal concentrations without algae were biased caused by contamination. Therefore, evaluation was only performed using the three highest treatments. Determination of the results was based on the geometric mean of the measured concentrations after 0 and 72 h in the 3 highest treatments. Inhibition of growth was recorded resulting in an ErC10 (72h) of 95 µg/L and an ErC50 (72h) of 160 µg/L. Inhibition of yield was recorded resulting in an EyC10 (72h) of 69 µg/L and an EyC50 (72 h) of 110 µg/L. The NOEC was observed to be at 82.6 µg/L for growth rate and yield and thus below the highest measured test concentration of 179.9 µg/L (based on geometric mean).