Butanal, reaction products with aniline

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: 100 μL, depending on the expected concentration).

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations:
To produce the different Water Accommodated Fractions (WAFs) 1.2, 3.1, 9.8, 31.8 and 100.1 mg of the test item were weighed out and were added each to 1 litre of dilution water. All solutions were treated with an ultra turrax for 60 seconds at 8000 rpm and afterwards stirred for 24 h on magnetic stirrer. Undissolved particles of the test item were removed by filtration using folded filters with a pore size of 7-12 μm and aseptic filters (sartobran 150 Sterile Capsule) with a pore size of 0.45 μm + 0.2 μm. The pH was measured to be 7.8 in all solutions.
To produce the different loading rates appropriate amounts of the prepared solutions were diluted with dilution water to a volume of 100 mL and 0.543 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each loading rate 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 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 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 : The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth 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

Test temperature:

21-24°C

pH:

7.9 at 0 hour
8.9 at 72 hour

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32 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 for measurements, which were not replaced.
- Experimental design: 5 test concentration 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: 1.0, 3.2, 10, 32 and 100 mg/L
- 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:

EL50

Effect conc.:

128 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 112-151 mg/L

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 17-26 mg/L

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 min

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The test item has not a definite or unique structure and consists of several constituents. A suitable selective and sensitive chromatographic method for the determination of the test item in aqueous solutions could not be established and no accompanying analysis by the means of a chromatographic method cannot be performed.
Because aniline seems to have an influence on the toxic effects of Butanal, reaction products with aniline the aniline content was analysed during the test with the sponsor’s agreement. To record the total amount of dissolved substance the content of the test item during the exposure period was additionally verified by DOC determination.
Water Accommodated Fractions (WAFs) were produced to test the effects at different loading rates and all results are expressed in terms of Effective Loadings (EL).

Analysis (DOC determination)

Test concentration [mg/L]	DOC values [mg/L] 0 hour	DOC values [mg/L] 72 hour
Control	n.d.	2.92*
10	5.63	7.87
100	13.5	13.2

Comments:

* In spite of filtration it is possible that algal cells were also measured after 72 hours exposure using particle DOC determination. Because of the limit of quantitation of 2 mg/L DOC the measurement of 3.2 mg/L and 1 mg/L test item concentration was not performed.

n.d. not determined

Analysis (HPLC value aniline)

Test concentration [mg/L]	HPLC values aniline [mg/L] 0 hour	HPLC values aniline [mg/L] 72 hour
Control	n.d.	< 0.026
1.0	0.2134	0.1949
10	1.082	1.029
100	4.302	4.101

Comments: n.d. not determined

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 the test item to alga (Desmodesmus subspicatus) was tested according to OECD guideline 201 ' Alga, Growth Inhibition Test' (2006). During 72 hours exposure an ELC50 of 128 mg/L and EL10 of 22 mg/L was measured and a NOEL of 3.2 mg/L was calculated.

Executive summary:

A study was performed to assess the adverse effects of the test item 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 range of concentrations, nominally 1.0, 3.2, 10, 32 and 100 mg/L of the test item dissolved in dilution water. Auxiliaries used to prepare the test media were an ultra turrax, magnetic stirrers, folded filters and aseptic filters.

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.

The following values were determined: ErL 50 (0-72 h): 128 mg/L, ErL 10 (0-72 h): 22 mg/L, NOEL [r] (tα 0.05): 3.2 mg/L and LOEL [r] (tα 0.05): 10 mg/L.

The test item has not a definite or unique structure and consists of several constituents. A suitable selective and sensitive chromatographic method for the determination of the test item in aqueous solutions could not be established and an accompanying analysis by the means of a chromatographic method cannot be performed. Because aniline seems to have an influence on the toxic effects of Butanal, reaction products with aniline, the aniline content was analysed during the test with the sponsor’s agreement. To record the total amount of dissolved substance the content of the test item during the exposure period was additionally verified by DOC determination. Water Accommodated Fractions (WAFs) were produced to test the effects at different loading rates and all results are expressed in terms of Effective Loadings (EL).

Description of key information

The acute toxicity of the test item to alga (Desmodesmus subspicatus) was tested according to OECD guideline 201 ' Alga, Growth Inhibition Test' (2006). During 72 hours exposure an ELC50 of 128 mg/L and EL10 of 22 mg/L was measured and a NOEL of 3.2 mg/L was calculated.

Key value for chemical safety assessment

EC50 for freshwater algae:

128 mg/L

EC10 or NOEC for freshwater algae:

22 mg/L

Additional information

The test item has not a definite or unique structure and consists of several constituents. A suitable selective and sensitive chromatographic method for the determination of the substance in aqueous solutions could not be established and analytical monitoring by means of a chromatographic method cannot be performed. As aniline seems to have an influence on the toxic effects of the test item, the aniline content was analysed during the test with the sponsor’s agreement. To record the total amount of dissolved substance the content of the substance during the exposure period was additionally verified by DOC determination. Water Accommodated Fractions (WAFs) were produced to test the effects at different loading rates and all results are expressed in terms of Effective Loadings (EL).