Dibutyl 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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Aliquots of the samples from the biological test were directly analysed by HPLC and UV/VIS-detection (range of the injection volume: 2-100 μL, depending on the expected concentration).

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations:
A stock solution was prepared to give the desired series of test concentrations. 100.3 mg of the test item were added to 1 litre of dilution water and treated for 1 h on a magnetic stirrer. The pH was measured to be 6.3.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.556 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 3 replicates were prepared. All flasks were sealed with cotton/cellulose stoppers.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

- Name: Desmodesmus subspicatus (formerly Scene-desmus subspicatus) Strain No. 86.81 SAG
- Sourca: 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 microcell counter, Sysmex F300, Digitana.
- 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:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21-24°C

pH:

at the start on the study: 6.8 -7.8
at the end of the study: 5.8 - 8.8

Nominal and measured concentrations:

nominal: 3.1, 6.3, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

Exposure conditions:
- Test vessels: 300 mL Erlenmeyer flasks with cotton/cellulose stoppers, test volume: 100 mL
- Culturing apparatus: 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.
- 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 microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced.
- Experimental design: 6 test concentrations plus 1 control, 3 replicates per concentration, 3 replicates per control, Initial cell density in the test cultures approximately 5000 cells per millilitre.
- Test item concentration/s: 3.1, 6.3, 12.5, 25, 50 and 100 mg/L
- Method of administration: stock solution
- 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.

Key result

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:

EC10

Effect conc.:

76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL = 65-83 %

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The lowest test item concentration 3.1 mg/L slightly dropped below the 80% recovery limit during the 72-hour exposure period. This is considered as not significant, because the highest determined test item concentration remained stable over the exposure time and therefore all results are expressed in terms of nominal concentrations. Effective concentrations ranged from 80.6 % to 103.0 % of nominal values at 0 hours, and from 79.6 % to 91.5 % of nominal values at 72 hours.

Analysis:

Test item concentration [mg/L]	HPLC values at 0 h [mg/L]	HPLC values at 72 h [mg/L]
Control	n.d.	< 0.0421
3.1	2.500	2.467
100	103.039	91.452

Validity criteria fulfilled:

yes

Remarks:

The factor of biomass parameter 16; -The mean of the replicate coefficients of variation in the section-by-section growth < 35%; - The coefficient of variation of the mean specific growth rate replicates in the control < 7%

Conclusions:

The acute toxicity of dibutyl hydrogen phosphate to alga (Desmodesmus subspicatus) was tested according to OECD guideline 201 ' Alga, Growth Inhibition Test'. During 72 hours exposure an ErC50 of >100 mg/L and ErC10 of 76 mg/L was received and a NOEC of 50 mg/L was calculated.

Executive summary:

A study was performed to assess the adverse effects of dibutyl hydrogen phosphate on the growth rate (= rate of increase in cell density with time) 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 range of concentrations, nominally 3.1, 6.3, 12.5, 25, 50 and 100 mg/L of dibutyl hydrogen phosphate dissolved in dilution water. Auxiliary used to prepare the test media was a magnetic stirrer. 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 Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment. During 72 hours exposure an ErC50 of >100 mg/L and ErC10 of 76 mg/L was measured and a NOEC of 50 mg/L was calculated. The lowest substance concentration 3.1 mg/L slightly dropped below the 80% recovery limit during the 72-hour exposure period. This is considered as not significant, because the highest determined test item concentration remained stable over the exposure time and therefore all results are expressed in terms of nominal concentrations. Effective concentrations ranged from 80.6 % to 103.0 % of nominal values at 0 hours, and from 79.6 % to 91.5 % of nominal values at 72 hours.

Description of key information

The acute toxicity of dibutyl hydrogen phosphate to alga (Desmodesmus subspicatus) was tested according to OECD guideline 201 ' Alga, Growth Inhibition Test'. During 72 hours exposure an ErC50 of >100 mg/L and ErC10 of 76 mg/L was received and a NOEC of 50 mg/L was calculated.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

76 mg/L

Additional information

Dibutyl hydrogen phosphate was assessed within the scope of the ICCA/HPV-program. Data on toxicity of dibutyl hydrogen phosphate towards aquatic plants was found for Selenastrum capricornutum (new name: Pseudokirchnerella subcapitata). The study was carried out at the National Institute of Technology and Evaluation, Japan following the national standard procedure which corresponds to the OECD Guideline 201. A 72h-EC50 of 92 mg/L and a 72h-NOEC of 100 mg/L was reported, relating to biomass. According to the Guidance on information requirements and chemical safety assessment R.7b (ECHA, 2012) results based on the biomass should not be used for assessment since biomass results depend on the growth rate of the species as well as the test duration and other elements of the test design. Thus, the presented result was scored as not reliable.

A new study was performed to assess the adverse effects of dibutyl hydrogen phosphate on the growth rate (= rate of increase in cell density with time) 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 range of concentrations, nominally 3.1, 6.3, 12.5, 25, 50 and 100 mg/L of dibutyl hydrogen phosphate dissolved in dilution water. Auxiliary used to prepare the test media was a magnetic stirrer. 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 Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment. During 72 hours exposure an ErC50 of >100 mg/L and ErC10 of 76 mg/L was measured and a NOEC of 50 mg/L was calculated. The lowest test item concentration 3.1 mg/L slightly dropped below the 80% recovery limit during the 72-hour exposure period. This is considered as not significant, because the highest determined substance concentration remained stable over the exposure time and therefore all results are expressed in terms of nominal concentrations. Effective concentrations ranged from 80.6 % to 103.0 % of nominal values at 0 hours, and from 79.6 % to 91.5 % of nominal values at 72 hours. This toxicity study is classified as acceptable and satisfies the guideline requirements for the study towards aquatic plants.