Hexanamide, N-(2-ethylhexyl)-3,5,5-trimethyl-

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:

2013-10-11 to 2013-10-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to geometric mean measured concentrations of 88.5 - 177 - 310 - 568 - 1381 µg test item/L.


- Sampling method:The concentrations of the test item were analytically determined from all test concentrations and the control at the start of the exposure (0 h) and at the end of the exposure (72 h). Samples at the start of the exposure were taken from an additional replicate without algae. At the end of the exposure samples were taken directly from the pooled test replicates.


- Sample storage conditions before analysis: All original and diluted samples were stored at room temperature until sample preparation and until analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:A stock solution with a nominal loading of 1 mL test item / L was prepared once with dilution water three days prior to start of the exposure. The test item was placed by pipetting on the surface of the dilution water.

A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for three days at approximately 23 °C. After a separation of the phases of 1 hour, the saturated solution was taken from the homogeneous phase and was used as stock solution.

- Eluate: Dilution water
- Differential loading: The saturated solution and further four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 % of the saturated solution, corresponding to geometric mean measured concentrations of 88.5 - 177 - 310 - 568 - 1381 µg test item/L.
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

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 every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+Mg/L.

Test temperature:

min.: 22.5 max.: 23.5, mean value: 23.0

pH:

Environmental Conditions

Geometric mean pH-value
measured test item
concentration
[µg/L]
Start; 0 hours End; 72 hours
1381 7.88 8.15
568 7.92 8.34
310 7.92 8.46
177 7.96 8.42
88.5 7.97 8.41
Control 8.03 8.27

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- 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: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4864 cells/mL
- Control end cells density: 1660533 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6



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, mean value: 74.8



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:
Results of the Range Finding Test (0-72 hours)
Saturated Solution
(Nominal)
[%] Growth Rate Inhibition [%] Yield Inhibition [%]
100 39 86
10.0 -3 -18
1.00 1 5

Negative inhibition = increase of growth

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

310 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

597 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: < 568 - 656

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

962 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

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

Current Study Valid Range (average ± 3 x SD)
Growth rate inhibition
ErC50 0.590 0.675 ± 0.440
95 % confidence interval 0.564 - 0.623
Yield inhibition
EyC50 0.299 0.313 ± 0.139
95 % confidence interval 0.278 - 0.324
SD = Standard deviation

Reported statistics and error estimates:

NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. The Equal Variance Test failed for growth rate. Growth rate values were transformed (y = y squared) to pass the Equal Variance Test.

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

Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[µg/L]	No.	0 hours	24 hours	48 hours	72 hours
1381	1	4864	<LOQ	<LOQ	10403
	2	4864	<LOQ	<LOQ	23128
	3	4864	<LOQ	<LOQ	15095
	Mean	4864	n.a.	n.a.	16209
568	1	4864	18219	115950	1111933
	2	4864	22405	153362	946114
	3	4864	25339	124686	1572001
	Mean	4864	21988	131333	1210016
310	1	4864	19626	153145	1672538
	2	4864	23361	205720	1569044
	3	4864	21081	239974	2002139
	Mean	4864	21356	199613	1747907
177	1	4864	29564	215340	1827942
	2	4864	26847	248661	1793576
	3	4864	41931	321856	1928675
	Mean	4864	32781	261952	1850064
88.5	1	4864	34870	285275	1784376
	2	4864	28092	309555	1900157
	3	4864	40222	347423	2171342
	Mean	4864	34395	314084	1951958
Control	1	4864	38557	294080	1734239
	2	4864	30612	244469	1650919
	3	4864	29771	232851	1542366
	4	4864	35958	240007	1547754
	5	4864	38429	248806	1634097
	6	4864	42539	291872	1883007
	Mean	4864	35978	258681	1665397

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1381	1		0.253	87		5539	100
	2		0.520	73		18264	99
	3		0.378	81		10231	99
	Mean	(+)	0.384	80	(+)	11345	99
568	1		1.81	7		1107069	33
	2		1.76	10		941250	43
	3		1.93	1		1567137	6
	Mean	(+)	1.83	6	(+)	1205152	27
310	1		1.95	0		1667674	0
	2		1.93	1		1564180	6
	3		2.01	-3		1997275	-20
	Mean	(-)	1.96	-1	(-)	1743043	-5
177	1		1.98	-2		1823078	-10
	2		1.97	-1		1788712	-8
	3		1.99	-3		1923811	-16
	Mean	(-)	1.98	-2	(-)	1845200	-11
88.5	1		1.97	-1		1779512	-7
	2		1.99	-2		1895293	-14
	3		2.03	-5		2166478	-30
	Mean	(-)	2.00	-3	(-)	1947094	-17
Control	1		1.96			1729375
	2		1.94			1646055
	3		1.92			1537502
	4		1.92			1542890
	5		1.94			1629233
	6		1.99			1878143
	Mean		1.95			1660533

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 310 µg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 962 (890 – 1046) µg/L and 743 (608 – 1029) µg/L, respectively.
All effect levels are given based on geometric mean measured concentrations of the test item.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) with the definitive exposure phase from2013-10-14 to 2013-10-17.The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4864 cells/mL. Based on a preliminary range findingtest a saturated solution with a nominal loading of 1 mL test item/Land further four dilution levels of the saturated solution weretestedin a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0 - 100 %, corresponding to geometric mean measured concentrations of 88.5 - 177 - 310 - 568 - 1381 µg test item/L.Three replicates were tested for each test item concentration and six replicates for the control.Environmental conditionswere determined to be within the acceptable limits.

The concentrations of the test item were analytically verified via LC-MS/MS at the start and the end of the exposure of all test concentrations and the control.
All effect values given are based on the geometric mean measured test item concentrations of the test item.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of test item (0-72 hours)

                   based on geometric mean measured test item concentrations [µg/L]

	Growth Rate Inhibition Geometric mean measured test item concentration [µg/L]
NOEC	310
LOEC	568
ErC10	597 (< 568 – 656)
ErC20	673 (607 – 732)
ErC50	962 (890 – 1046)
	Inhibition of Yield Geometric mean measured test item concentration [µg/L]
NOEC	310
LOEC	568
EyC10	437 (312 – 539)
EyC20	514 (402 – 700)
EyC50	743 (608 – 1029)

Description of key information

In this study the test item was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 310 µg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 962 (890 – 1046) µg/L and 743 (608 – 1029) µg/L, respectively.

All effect levels are given based on geometric mean measured concentrations of the test item.

Key value for chemical safety assessment

EC50 for freshwater algae:

962 µg/L

EC10 or NOEC for freshwater algae:

597 µg/L