Octanoic acid, compound with octylamine (1:1)

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:

2016-05-09 to 2016-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Octanoic acid, compound with octylamine (1:1)

EC Number:

241-477-7

EC Name:

Octanoic acid, compound with octylamine (1:1)

Cas Number:

17463-34-2

Molecular formula:

C8H19N.C8H16O2

IUPAC Name:

octan-1-amine; octanoic acid

Test material form:

other: Pale brown, waxy solid

Details on test material:

Test Item: Caprylamine caprylate

Chemical name: Octanoic acid, compound with octylamine

CAS number: 17463-34-2

Molecular formula: C8H19N.C8H16O2

Molecular weight: 273.5 g/mol

Appearance: Pale brown, waxy solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution
100 mg test item/L were freshly prepared with dilution water.

Dispersion treatment
Agitation until the solution is visually clear

Test concentrations
5 concentrations were tested in a geometrical series with a dilution factor of √10: 0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L.
The concentrations were based on the results of a preliminary range finding test (non-GLP)

Control
Six replicates (without test item) were exposed under the same conditions as the test concentrations.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 23.0 max.: 24.0 mean value: 23.5

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
10.0 8.09 8.19
3.16 8.13 8.24
1.00 8.01 9.01
0.316 8.06 9.41
0.100 8.02 9.48
Control 8.12 9.52

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

The measured concentrations of Caprylamine caprylate in the fresh media (0 h) were in the range of 114 to 120 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 82 to 115 % of the nominal values of all concentrations. Therefore, the nominal concentrations of the test item were used for the calculations.

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 5 x 10^3 - 10^4 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 2289490 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:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 4440 - 8880 lux, mean value: 5518 lux,



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.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.316 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI 0.476 - 0.865

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.835 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI 0.626 - 0.929

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI 1.06 - 1.65

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.316 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI <0.100 - 0.301

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.303 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI 0.237 - 0.384

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.504 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: CI 0.417 - 0.625

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
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-04-26 to 2016-04-29 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.460 0.780 ± 0.473
95 % confidence interval 0.443 – 0.481
Yield inhibition
EyC50 0.317 0.411 ± 0.276
95 % confidence interval 0.208 – 0.347
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC- and LOEC-values
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield. The following statistical tests were conducted:
Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01.
Levene’s test on variance homogeneity was done with a significance level 0.01.
Monotonicity was done by trend analysis by contrasts (significance level 0.05).
William’s multiple sequential t-test procedure was done with a significance level 0.05.
Welsh-t-test after Bonferroni-Holm was done with a significance level of 0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- ToxRat, ToxRat Solutions GmbH
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

Cell Densities

Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours		48 hours		72 hours
10.0		1		6305		2787		<LOQ		<LOQ
		2		6305		2852		<LOQ		<LOQ
		3		6305		3325		<LOQ		<LOQ
		Mean		6305		2988		n.a.		n.a.
3.16		1		6305		9116		3872		<LOQ
		2		6305		9668		3503		<LOQ
		3		6305		9138		3365		<LOQ
		Mean		6305		9307		3580		n.a.
1.00		1		6305		28547		56951		197151
		2		6305		28068		80563		383899
		3		6305		31391		67779		318233
		Mean		6305		29335		68431		299761
0.316		1		6305		27391		301776		1795874
		2		6305		25984		316559		2195664
		3		6305		34749		219562		1375448
		Mean		6305		29375		279299		1788995
0.100		1		6305		33201		418826		2151632
		2		6305		33060		439320		2346312
		3		6305		29770		406028		2206940
		Mean		6305		32010		421391		2234961
Control		1		6305		29240		315882		2280460
		2		6305		44159		480652		2251650
		3		6305		36761		435875		2304478
		4		6305		29796		353414		2206320
		5		6305		40714		428805		2172774
		6		6305		43730		492487		2521259
		Mean		6305		37400		417853		2289490

< LOQ = below the limit of quantification (2736 cells/mL)                            n.a. = data not determinable

 

 Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Nominal test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
10.0	1		n.a.	100		n.a	100
	2		n.a	100		n.a	100
	3		n.a	100		n.a	100
	Mean	(+)	n.a	100	(+)	n.a	100
3.16	1		n.a	100		n.a	100
	2		n.a	100		n.a	100
	3		n.a	100		n.a	100
	Mean	(+)	n.a	100	(+)	n.a	100
1.00	1		1.15	42		190846	92
	2		1.37	30		377594	83
	3		1.31	33		311928	86
	Mean	(+)	1.28	35	(+)	293456	87
0.316	1		1.88	4		1789569	22
	2		1.95	1		2189359	4
	3		1.80	9		1369143	40
	Mean	(-)	1.88	4	(+)	1782690	22
0.100	1		1.94	1		2145327	6
	2		1.97	0		2340007	-2
	3		1.95	1		2200635	4
	Mean	(-)	1.96	0	(-)	2228656	2
Control	1		1.96			2274155
	2		1.96			2245345
	3		1.97			2298173
	4		1.95			2200015
	5		1.95			2166469
	6		2.00			2514954
	Mean		1.97			2283185

 Section-by-Section and Average Specific Growth Ratesof the Control Group (0 - 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.53	2.38	1.98	1.96	0.423	21.5	21.4
	2	1.95	2.39	1.54	1.96	0.422	21.5
	3	1.76	2.47	1.67	1.97	0.441	22.4
	4	1.55	2.47	1.83	1.95	0.472	24.2
	5	1.87	2.35	1.62	1.95	0.373	19.1
	6	1.94	2.42	1.63	2.00	0.398	19.9
				Mean	1.97
				SD ±	0.02
				CV [%]	0.89

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Caprylamine caprylate was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test item concentrations): The NOEC (0-72 h) for inhibition of growth and inhibition of yield was 0.316 and 0.100 mg/L. The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 1.15 (1.06 – 1.65) mg/L and 0.504 (0.417 – 0.625) mg/L, respectively. All effect values are based on the nominal concentrations of Caprylamine caprylate.

Executive summary:

The toxicity of Caprylamine caprylate to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3 from 2016-05-09 to 2016-05-12 at the test facility. The aim of the study was the determination of NOEC, LOEC, EC₁₀- , EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A stock solution of 100 mg/L was freshly prepared with dilution water. Five concnetrations were tested in a geometrical series with a dilutionfactor of √10:0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L. The environmental conditions were within the acceptable limits.

 

The test media were visually clear throughout the test period. The concentrations of the test item Caprylamine caprylate in all test concentrations and the control were analytically verified by LC-MS/MS at the start and the end of exposure.
The measured concentrations of Caprylamine caprylate in the fresh media (0 h) were in the range of 114 to 120 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 82 to 115 % of the nominal values of all concentrations.

Therefore, all effect values given are based on the nominal test item concentrations of Caprylamine caprylate.

 

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

  based on nominal test item concentrations [mg/L]

	Growth RateInhibition nominal test item concentrations [mg/L]
NOEC	0.316
LOEC	1.00
ErC10	0.680 (0.476 – 0.865)
ErC20	0.835 (0.626 – 0.929)
ErC50	1.15 (1.06 – 1.65)
	Inhibition of Yield nominal test item concentrations [mg/L]
NOEC	0.100
LOEC	0.316
EyC10	0.220 (<0.100 – 0.301)
EyC20	0.303 (0.237 – 0.384)
EyC50	0.504 (0.417 – 0.625)