3,5,5-trimethylhexanal

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-01-06 to 2009-01-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study conducted according to GLP and in compliance with agreed protocols, with minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006)

Deviations:

no

Principles of method if other than guideline:

The dilution level of the test substance were analysed by DOC analysis.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,5,5-trimethylhexanal

EC Number:

226-603-0

EC Name:

3,5,5-trimethylhexanal

Cas Number:

5435-64-3

Molecular formula:

C9H18O

IUPAC Name:

3,5,5-trimethylhexanal

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Saturated solution prepared with 100 mg/L, the saturated solution and five further dilution levels were tested, nominal: 0.32 - 1.00 - 3.20 - 10.0 - 32.0 % of the saturated solution
- Sampling method: Separate replicates without algae, incubated as the test replicates
- Sample storage conditions before analysis: immediate analysis out of additional replicates

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of a saturated solution acc. to OECD Series, No. 23 (2000). 100 mg/L of the test item were directly weighed out, freshly prepared with dilution water and shaken with 20 rpm for 24 h at room temperature in a brown glass flask. After a separation phase of 2 h the saturated solution was taken from the homogeneous liquid phase.
- Differential loading: nominal: 0.32 - 1.00 - 3.20 - 10.0 - 32.0 - 100 % of the saturated solution
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No undissolved material visible.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 h of exposure, 0.5 mL aliquots of the test medium (i.e. samples of exposed algal cells) from the geometric mean measured concentration of 47.6 mg/L and the control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 3 days under test conditions.

Test conditions

Hardness:

Test medium (nominal): 0.24 mmol CaMg/L

Test temperature:

Min: 21 °C, Max: 23 °C, Mean: 22 °C

pH:

Geometric mean measured test item concentrations
[mg/L] pH-value
Start; 0 h End; 72 h
47.6 7.61 7.69
15.1 7.62 7.95
4.68 7.74 8.19
1.19 7.70 8.30
0.98 7.71 8.15
0.32* 7.83 8.09
Control 8.29 8.35

Dissolved oxygen:

not measured

Salinity:

freshwater conditions

Nominal and measured concentrations:

nominal: 0.32 - 1.00 - 3.20 - 10.0 - 32.0 - 100.0 % of the saturated solution
gemoetric mean measured: 0.32* - 0.98 - 1.19 - 4.68 - 15.1 - 47.6 mg/L
*: nominal dilution level of the saturated solution, not regarded in statistical analysis

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 4878 cells/mL
- Control end cells density: Mean 553814 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Range finding study
- Test concentrations: 1 - 100
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Remarks on result:

other: (26.7 - 40.1)

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 47.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

saturated solution

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
EC50 Values of the Reference Item based on nominal concentrations (mg/L), (0-72 h)

Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.69 0.828 ± 0.399
95 % confidence interval 0.64 – 0.74
Yield inhibition
EyC50 0.31 0.352 ± 0.143
95 % confidence interval 0.30 – 0.32

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

Any other information on results incl. tables

EC-Values and 95 % Confidence Intervals of 3,5,5-Trimethylhexanal(0-72 h)

calculated concentrations based on the geometric mean measured DOC content of the test item [mg/L]

                               

EC-Values	Rate-related inhibition
ErC10	30.4 (23.1 – 38.1)
ErC20	44.6 (36.8 – 47.6)
ErC50	> 47.6*
	Yield inhibition
EyC10	16.8 (13.8 – 21.0)
EyC20	20.0 (16.4 – 24.5)
EyC50	33.0 (26.7 – 40.1)

* = saturated solution

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
47.6		1		4878		7896	42393	201779
		2		4878		7158	36844	203870
		3		4878		8610	34216	171821
		Mean		4878		7888	37818	192490
15.1		1		4878		21717	148584	498286
		2		4878		21079	146932	604843
		3		4878		21410	146307	478368
		Mean		4878		21402	147274	527166
4.68		1		4878		23168	174294	673457
		2		4878		21481	157299	593958
		3		4878		23050	159756	684266
		Mean		4878		22566	163783	650560
1.19		1		4878		23501	153027	649792
		2		4878		20708	144032	664991
		3		4878		21661	163281	712919
		Mean		4878		21957	153447	675901
0.98		1		4878		20481	153065	688452
		2		4878		23861	134082	613044
		3		4878		22121	153127	629039
		Mean		4878		22154	146758	643512
0.32*		1		4878		21472	160951	799071
		2		4878		22537	127533	701119
		3		4878		23082	162414	707990
		Mean		4878		22364	150299	736060
Control		1		4878		16271	119660	470795
		2		4878		22091	132923	668094
		3		4878		19534	146006	622452
		4		4878		19717	117329	520783
		5		4878		24206	125713	520688
		6		4878		20029	145012	520069
		Mean		4878		20308	131107	553814

             * = nominal dilution level of the saturated solution [%], not regarded in statistical analysis

Evaluation after 72 h

Geometric mean measured test item concentration	Replicate	Yield		Inhibition of yield	Growth rate		Rate-related inhibition
[mg/L]	No.	[cells/mL]		[%]	[d^-1]		[%]
47.6	1		196901	64.13		1.241	21.21
	2		198992	63.75		1.244	21.00
	3		166943	69.59		1.187	24.62
	Mean		187612	65.82		1.224	22.28
15.1	1		493408	10.12		1.542	2.08
	2		599965	-9.30		1.607	-2.02
	3		473490	13.74		1.529	2.94
	Mean		522288	4.85		1.559	1.00
4.68	1		668579	-21.80		1.643	-4.30
	2		589080	-7.31		1.601	-1.64
	3		679388	-23.76		1.648	-4.63
	Mean		645682	-17.62		1.630	-3.52
1.19	1		644914	-17.48		1.631	-3.54
	2		660113	-20.25		1.638	-4.03
	3		708041	-28.98		1.662	-5.50
	Mean		671023	-22.24		1.644	-4.36
0.98	1		683574	-24.53		1.650	-4.76
	2		608166	-10.79		1.611	-2.31
	3		624161	-13.70		1.620	-2.85
	Mean		638634	-16.34		1.627	-3.31
0.32*	1		794193	-44.68		1.700	-7.91
	2		696241	-26.83		1.656	-5.15
	3		703112	-28.09		1.659	-5.35
	Mean		731182	-33.20		1.672	-6.14
Control	1		465917			1.523
	2		663216			1.640
	3		617574			1.616
	4		515905			1.557
	5		515810			1.557
	6		515191			1.556
	Mean		548936			1.575

negative inhibitions = increase of growth
* = nominal dilution level of the saturated solution [%], not regarded in statistical analysis

Section-by-Section and Average Specific Growth Rates of the Control Group

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD [±]	CV [%]	Mean CV [%]
		section-by-section
		0 – 24 h	24 – 48 h	48 – 72 h
Control	1	1.205	1.995	1.370	1.523	0.417	27.4	17.0
	2	1.510	1.795	1.615	1.640	0.144	8.77
	3	1.387	2.011	1.450	1.616	0.344	21.3
	4	1.397	1.784	1.490	1.557	0.202	13.0
	5	1.602	1.647	1.421	1.557	0.120	7.69
	6	1.412	1.980	1.277	1.556	0.373	23.9
				Mean	1.575
				SD [±]	0.04
				CV [%]	2.78

                                                SD = Standard deviation      CV = percent coefficient of variation

Recovery of Alga Cells

Geometric mean measured test item concentration [mg/L]	Incubation time                    [d]	Cell density [cells/mL]		Growth rate after incubation time [d^-1]
		Initial^#	Final
47.6	3	9625	1118190	1.59
			1263413	1.63
			1318961	1.64
Control		27691	1168930	1.25
			1321737	1.29

^#) calculated from the cell density of the definitive test after 72 h

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

n this study 3,5,5-Trimethylhexanal was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (geometric mean measured concentrations of the test item): The EC50-values with 95 % confidence intervals for specific growth rate (ErC50) and yield (EyC50) after 72 h were > 47.6 mg/L (> saturated solution of 100 mg/L) and 33.0 (26.7 – 40.1) mg/L, respectively.
All effect levels are given based on geometric mean measured concentrations of 3,5,5-Trimethylhexanal calculated from the DOC content in the dilution levels 1.0 – 3.2 – 10 – 32 – 100 %, corresponding to 0.98 – 1.19 – 4.68 – 15.1 and 47.6 mg 3,5,5-Trimethylhexanal/L. The DOC content of the dilution level 0.32 % was not determinable due to low content. However this dilution level showed no significant effects and was not considered in statistical analysis.

Executive summary:

The toxicity of 3,5,5-Trimethylhexanal the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. The aim of the study was to assess the effects on growth rate and yield production over a period of 72 h.
The study was conducted under static conditions with an initial cell density of approximately 2 - 5 x 10³ cells/mL. A saturated solution (The maximum dissolved concentration of the test item that can be achieced under the test conditions in the test medium, acc. to OECD Series, No 23 (2000)6.) of 100 mg/L was prepared. The saturated solution and further five dilution levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.32 - 1.0 - 3.2 - 10 - 32 and 100 % of the saturated solution. Three replicates were tested for the test item dilution levels and six replicates for the control.
The dilution levels of 3,5,5-Trimethylhexanal were analysed at test start and end via DOC-analysis. All effect levels are given based on geometric mean measured concentrations of the test item calculated from the DOC content in the dilution levels 1.0 – 3.2 – 10 – 32 – 100 %, corresponding to 0.98 – 1.19 – 4.68 – 15.1 and 47.6 mg test item/L . The DOC content of the dilution level 0.32 % was not determinable due to low content. However this dilution level showed no significant effects and was not considered in statistical analysis.

After 72 h algae were transferred from the geometric mean measured concentration of 47.6 mg/L and the control to fresh untreated medium and allowed to grow for further 3 d under test conditions. The test item effect was observed to be reversible. Therefore, there is potential for recovery following exposure up to the saturated solution.

EC-Values and 95 % Confidence Intervals of 3,5,5-Trimethylhexanal(0-72 h)

calculated concentrations based on the geometric mean measured DOC
content of the test item [mg/L]

                               

EC-Values	Rate-related inhibition
ErC10	30.4 (23.1 – 38.1)
ErC20	44.6 (36.8 – 47.6)
ErC50	> 47.6*
	Yield inhibition
EyC10	16.8 (13.8 – 21.0)
EyC20	20.0 (16.4 – 24.5)
EyC50	33.0 (26.7 – 40.1)

* = saturated solution