Tris(2-ethylhexyl) phosphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 November 2009 to 22 January 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met. Non-standard methods were used in order to obtain the maximum achievable concentration yielding measured concentrations in excess of water solubility, therefore the endpoint should be used with caution but is still considered in the weight-of-evidence approach for this endpoint & subsequent hazard assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Analytical monitoring:

yes

Details on sampling:

The stability of the test concentrations during exposure were examined by gas chromatography at 0, 24, 48 and 72 hours.

Vehicle:

no

Details on test solutions:

To accelerate the solution procedure a five fold amount of the water solubility of the test item were taken to produce the stock solution (water solubility: < 1 mg/L (according to data from the sponsor). To achieve this 5.1 mg/L of the test item were added to 1 litre of dilution water, treated for 60 seconds at 8000 rpm. with an ultra turrax and treated for 24 h on a magnetic stirrer. Finally undissolved particles of the test item were removed by centrifuging the stock solution for 20 minutes at 9000 rpm and decanting the supernatant. The pH was measured to be pH 6.1.
To produce the different test item concentrations the prepared stock solution was diluted with dilution water to a volume of 40 mL. Afterwards 5 mL of nutrient medium and 5 mL of algal inoculum with a cell density of 5000 cells/mL were added resulting in the final nominal concentrations. For each test item concentration and control 3 replicates were prepared.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The organisms were Desmodesmus subspicatus, strain no. 86.81 SAG. The organisms were sourced from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
Exponentially-growing stock cultures were maintained in the test facility under constant temperature conditions (21-24°C with a maximum fluctuation of +/- 2°C) at a light intensity in the range 60 - 120 µE. x m-² x s-¹ (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell- counter, Sysmex F300, Digitana.
Pre-cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different nutrient medium. The algal inocula for a test were taken from an exponentially-growing pre-culture and were mixed with the nutrient medium (annex 1) to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not examined.

Hardness:

No data available

Test temperature:

21-24°C

pH:

pH at test start was 8.2

Dissolved oxygen:

No data available

Salinity:

No data available.

Nominal and measured concentrations:

Nominal concentrations: 0.409, 0.769 and 1.499 mg/L.

Details on test conditions:

The cell density at test start was 5000 cells/mL. Test concentrations and controls were tested in triplicate.
The test vessels were 300 mL Erlenmeyer flasks with cotton stoppers. The algae were cultured for 72 hours in a light chamber in which a temperature in the range 21°C to 24°C was maintained at +/- 2°C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily in a water filled flask which was incubated under the same conditions as the test flasks.
At the average of the test solutions, a light intensity in the range 60 to 120 µE. x m-² x s-¹, or an equivalent range of 4000 to 8000 lx, was used. Light intensity was checked before start of the study.
The cell densities were measured at 24 hour intervals.
In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, test flasks at an additional higher test concentration without algae were run in parallel to the geometric series of test concentrations.
The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r], respectively, of the algal population.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.876 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.876 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.876 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: determined according to Williams Multiple Sequential t-test procedure

Details on results:

No toxic effects against algae were observed at the limit of water of solubility under exposure conditions. Controls showed exponential growth during the study, and the coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was 1.5%. The factor of the biomass parameter, measured in the control between 0 and 72 h was 68.7.
Effective concentrations (expressed in terms of geometric mean measured concentrations) correspond to 100% of nominal values at 0 hours, ranged from 70.4-87.5% at 24 hours; from 14.7-66.4% at 48 hours and from 21.6-38.2% of nominal values at 72 hours, respectively.
pH at the start of the study was 8.2 in all groups, and at 72 h was 8.0, 8.0, 7.9 and 7.8 in the control, 0.409, 0.769 and 1.499 mg/L groups, respectively.
Test concentration analyses are shown in Table 1.

Results with reference substance (positive control):

Not examined.

Reported statistics and error estimates:

The NOEC was determined according to Williams Multiple Sequential t-Test Procedure. EC50 and EC10 values were calculated by probit analysis.

Table 1. Test concentration Analysis

Test item concentration (mg/L)	GC values (mg/L)
	0 h	24 h	48 h	72 h
Control	< 0.0546	< 0.0546	< 0.0546	< 0.0546
0.409	0.409	0.288	0.060	0.122^a
0.769	0.769	0.585	0.364	0.166
1.499	1.499	1.178	0.996	0.573
1.660 without algae	1.660	1.456	1.059	0.397

 ^aThe test item concentration 0.409 mg/L was calculated (after 72 h) based on the mean recovery rate of the test item concentrations 0.769 and 1.499 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

No toxic effects against algae were observed at the limit of water of solubility under exposure conditions.

Executive summary:

The study was performed to assess the adverse effects of Disflamoll TOF on the growth rate of the planktonic freshwater algal species Desmodesmus subspicatus over several generations. Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 0.409, 0.769 and 1.499 mg/L of Disflamoll TOF dissolved in water. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to Williams Multiple Sequential t-Test Procedure. No toxic effects against algae were observed at the limit of water of solubility under exposure conditions. The 72 h EC50 was calculated to be > 0.876 mg/L, whilst the NOEC was ≥ 0.876 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1998-03-16 till 1998-03-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted in accordance with international guidelines and in accordance with GLP. There were some minor deviations from standard guideline methodology but were not anticipated to affect the validity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Only 3 control replicates were used instead of 6

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling schedule: Concentrations of 0.30, 1.0, 3.0, 10, 20 and 40 mg/L and a concentration of 0 mg/L (control) were measured at 0, 24, 48 and 72 hours.

Vehicle:

yes

Details on test solutions:

Test solutions were made by weighing 50 mg of test substance and mixing into 125 mg of dispersant (HCO-40). This solution was diluted in culture medium and made up to 50 mL. This resulted in a stock solution of 1 000 mg/L. At the same time a dispersent only stock was also made (to 2 500 mg/L). Each test vessel was loadded with 96 mL of medium, and to this 4 mL of the stock solutions were added. This led to a test concentration and disperant control concentraion of 40 and 100 mg/L respectively.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The unicellular algae Selenastrum capricornutum was obtained from the American Type Culture Collection and was strain ATCC22662 on the 20 June 1996. The culture has been continually maintained and subcultured under aeptic condition at the Yokohama Laboratory facility. Reference substances are regularly to test algae sensitivity. The 72-hour test on growth inhibiton for potassium dichromate was 0.41 mg/L (determined by linear regresion.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Moderately hard synthetic freshwater

Test temperature:

22.8 - 24.2 °C

pH:

8.0 at the beginn of the test
9.6 - 10.1 at the end of the test

Nominal and measured concentrations:

Concentration: upto 40 mg/L (36.9 mg/L) plus blank control.

Details on test conditions:

In a prelimenary test algae was exposed to 0.3-40 mg/L (6 concentations, 1 replicate per concentration) of tris(2ethylhexyl) phospahte under the described testing conditions. Based on the test results the definitive test was conducted on 0 and 40 mg/L tris(2-ethylhexyl) phosphate, using 3 replicates for each.

During the test algae was shaken continously at 100 rpm under continuous illumination (4 000 - 5 000 lux). The starting density for the test was 1 x 10^4 cells/mL. Test volume was 100 mL, and medium used was as described by the OECD 201 guideline (OECD medium). 0.5 mL of test solution from each testing vessel was taken and diluted with 9.5 mL of electrolyte solution, the cells were then counted was conducted on a particle counter (Toa Medical Electronics Co., Ltd.). A seperate test vessel was set up to measure pH at each concentraion at test initiation and pH of one vessel of the 3 replicate test vessel was tested at the end of the test. A growth curve was created by plotting mean cell concentrations over time for each test concentration from which growth inhibition rate. NOEC and ECb50 were determined and estimated, respectively.

Chemical analysis was conducted on a gass chromotograher.

Reference substance (positive control):

yes

Remarks:

potaaium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: α: 0.05

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72

Dose descriptor:

EC50

Effect conc.:

> 40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: α: 0.05

Details on results:

Over 72 hours pH increased from 8.0 to 9.6 in the control and 10.1 in both the solvent and test substance exposures. Temperature remained between 22.8-24.2 °C. Measured concentration was 92 % of nominal over 72 hours and therefore nominal was used in the result report as per the guidelines.

The EC50 values and associated 95 % confidence limits could not be estimated as only one concentration was tested. However based on the response seen, it can be concluded that the EC50 for all endpoints is > 40 mg/L

The NOEC values were determined by Student’s t-test, subsequent to F test for homogeneity of variances. Statistical analyses were performed using Yukms Statlight #3 software (Yukms Corp., Tokyo) the significance level was set to α= 0.05.

Results with reference substance (positive control):

EC50 = 0.41 mg/L

Reported statistics and error estimates:

The no-observed-effect concentration (NOEC) was taken to be the concentration at which there was no significant difference with the dispersant control.

pH increases by more than 1 during the test, although not desirable this does not invalidate the results and is not part of the OECD 201 validity criterion. This has had no impact on the algal health.

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EbC50 and ErC50 were estimated to be > 40 mg/L, the NOEC was determined to be > 40 mg/L.

Executive summary:

The toxicity to algae was determinated according to the OECD method 201 for a period of 72 hours. The 72 hour EbC50 and ErC50 were estimated to be > 40 mg/L, the NOEC was determined to be > 40 mg/L.

Description of key information

ErC50 (Desmodesmus subspicatus); >0.876 mg/L; OECD 201; Weyers (2010)

ErC50 (Pseudokirchneriella subcapita); > 40 mg/L; OECD 201; Anon (1998)

Additional information

The toxicity to aquatic algae and cyanobacteria of the substance was assessed using a weight-of-evidence approach. Two key studies conducted in accordance with OECD 201 showed that the substance is not toxic to freshwater algae at, or above, the limit of water solubility.

Weyers (2010) exposed algal cultures to a series of test concentrations, employing non-standard methods for achieving the maximum concentration in algal test medium, all in excess of water solubility. Although the measured concentrations were in excess of the water solubility of the test item, the study indicates that exposure at nominal concentrations of 1.5 mg/L (geometric mean measured = 0.876 mg/L) had no adverse effects on the growth rate or yield of freshwater algae.

Anon (1998) exposed algal cultures to a series of test concentrations up to 40 mg/L, utilising a dispersant to enhance solubility in the test medium and obtain a maximum achievable concentration. No adverse effects on growth rate were observed at any concentration tested and the resulting NOEC was ≥40 mg/L.

As the substance displayed no adverse effects to freshwater algae at, or above, the limit of water solubility the substance was concluded as not toxic to aquatic algae and cyanobacteria and no hazard has been identified for this endpoint in accordance with REACH Annex I (section 3).