Diisobutyl hydrogen 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:

2020

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations
To produce the only test item concentration 200.1 mg of the test item were added to 2 litres of dilution water, treated for 60 seconds at 8000 rpm with an ultra turrax and stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 - 12 . The pH was measured to be 7.4.
100 mL of the solution were taken and 0.632 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 6 replicates were prepared. All flasks were sealed with cotton stoppers.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Name : Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG

Source : Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).

Maintenance and Acclimatisation : 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 to 120 µE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a particle counter, Z2, Beckmann Coulter.

Preparation of pre cultures : 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 growth medium.

Test cultures : The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

21 °C to 24 °C was maintained at +/- 2 °C

pH:

7

Nominal and measured concentrations:

100 mg/L (nominal)

Details on test conditions:

8.6 Exposure conditions
Test vessels : 300 mL Erlenmeyer flasks with cotton stoppers
test volume: 100 mL

Culturing apparatus : Shaking incubator / 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.

Light intensity : A light intensity ranging from 60 to
120 µE x m-2 x s-1, or an equivalent range of
4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.

Cell density
measurements : Cell densities were measured in a particle counter (Z2, Beckmann Coulter) by taking aliquots from each test flask, which were not replaced.


Experimental design : 1 test concentration plus 1 control

6 replicates per concentration, 6 replicates per control

Initial cell density in the test cultures approximately 5000 cells per millilitre.

Additionally test concentration without algae.

Test item
concentration/s : 100 mg/L (nominal)

Method of
administration : direct weighing

Duration of exposure : 72 hours

Criteria of effects : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

The validity criteria of the test were met:

The cell density in the control cultures increased by a factor of at least 16 within 72 hours.

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 did not exceed 35%.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 %.

 

Validity criteria fulfilled:

yes

Conclusions:

No toxic effects against algae were observed at a limit test concentration of 100 mg/L under exposure conditions.

Executive summary:

A study was performed to assess the adverse effects of Diisobutyl phosphate on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC)
No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of Diisobutyl phosphate dissolved in dilution water. Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer and a folded filter.

 

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 (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to STUDENT-t test for Homogeneous Variances. The following values were determined:

Results [mg/L]:

E_rC 50* (0-72 h): > 100

E_rC 10* (0-72 h): > 100

NOEC [r]* (t_α　0.05): ≥ 100

LOEC [r]* (t_α　0.05): > 100

No toxic effects against algae were observed at a limit test concentration of 100　mg/L under exposure conditions.

* Reduction of growth rate (E_rCx, NOEC　[r]) is the preferred endpoint according to OECD　201 and for regulatory purposes in the EU. Results relating to yield (E_yCx, NOEC　[y]) were calculated to fulfil regulatory requirements in some countries (but not in the　EU) and are given in the results section of this report.

The results are expressed in terms of nominal concentrations. Effective concentrations ranged from 100 % to 102 % of nominal values at 0　hours, from 103% to 107% of nominal values at 24　hours, from 103 % to 118 % of nominal values at 48　hours and from 101% to 114 % of nominal values at 72 hours.

 

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

should read >= 100 mg/L