N-acetylhexanelactam

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:

from 2017-11-20 to 2017-11-23 at the test facility

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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

Test material

Test material information

Constituent 1

Reference substance name:

N-acetylhexanelactam

EC Number:

217-565-6

EC Name:

N-acetylhexanelactam

Cas Number:

1888-91-1

Molecular formula:

C8H13NO2

IUPAC Name:

1-acetylazepan-2-one

Test material form:

liquid

Details on test material:

Batch: not yet known

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae. The samples were analysed with an HPLC-DAD method.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution, media preparation
100 mg test item/L was freshly prepared with dilution water (adjusted to pH 6.95). The dispersion was shaken until the solution was visually clear.

Test concentration
One limit concentration of 100 mg/L test item was tested. The concentration level is based on the results of a preliminary range finding test.
 
Control
Six replicates (without test item) were tested under the same test conditions as the test item replicates.

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): A three day old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5

pH:

Geometric mean
measured test
item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
78.7 6.91 8.26
Control 6.94 8.34

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

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 10^3 cells/mL, Actual: mean 6839 cells/mL
- Control end cells density: Mean 2799975 cells/mL
- No. of vessels per concentration (replicates): 6
- 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 0.064a2EDTA 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

This medium had a nominal hardness of 0.24 mmol Ca+Mg/L. The pH value was adjusted to 6.95 with 1 M HCl and 1 M NaOH one day before the experimental starting. The pH value was checked at the experimental starting and doesn’t has to adjusted again.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5956 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the saturated solution. A separate replicate of each test item concentration were prepared without algae and treated like the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition oy Yield

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 78.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

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: The limit concentration was visually clear throughout the exposure period.
- 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 2017-10-10 to 2017-10-13. 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 0.535 0.745 ± 0.524
95 % confidence interval 0.523 – 0.547
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95 % confidence interval 0.249 – 0.339
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

NOEC and LOEC values
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEC/LOEC calculations. When running a t-test, a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. 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.

Software
The data for the tables in this report are computer-generated and have been rounded for presentation from the full 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.

Any other information on results incl. tables

Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
78.7	1	6839	32219	463096	2420239
	2	6839	27535	412734	2711786
	3	6839	30004	430699	2529086
	4	6839	31256	355782	2546291
	5	6839	31025	458812	2226304
	6	6839	28977	438424	2816244
	Mean	6839	30169	426591	2541658
Control	1	6839	27772	350398	2768140
	2	6839	33565	381718	2856213
	3	6839	27125	320214	2259953
	4	6839	40772	518994	3068875
	5	6839	33542	465015	3246483
	6	6839	30235	391257	2600188
	Mean	6839	32169	404599	2799975

 

 

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
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
78.7	1		1.96	2		2413400	14
	2		1.99	0		2704947	3
	3		1.97	2		2522247	10
	4		1.97	1		2539452	9
	5		1.93	4		2219465	21
	6		2.01	0		2809405	-1
	Mean	(-)	1.97	2	(-)	2534819	9
Control	1		2.00			2761301
	2		2.01			2849374
	3		1.93			2253114
	4		2.04			3062036
	5		2.05			3239644
	6		1.98			2593349
	Mean		2.00			2793136

Negative inhibition = increase of growth

 

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	1.40	2.54	2.07	2.00	0.570	28.5	25.4
	2	1.59	2.43	2.01	2.01	0.420	20.9
	3	1.38	2.47	1.95	1.93	0.546	28.2
	4	1.79	2.54	1.78	2.04	0.440	21.6
	5	1.59	2.63	1.94	2.05	0.528	25.7
	6	1.49	2.56	1.89	1.98	0.542	27.4
				Mean	2.00
				SD ±	0.04
				CV [%]	2.13

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, no inhibition of growth on the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test item N-acetylhexanelactam. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 78.7 mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 78.7 mg/L, respectively.
All effect values given are based on geometric mean measured test item concentrations of N-acetylhexanelactam.

Executive summary:

The toxicity of N-acetylhexanelactam to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from2017-11-20 to 2017-11-23 at the test facility.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 6839 cells/mL. A stock solutionwith a nominal

loading of 100 mg test item/L was tested. Six replicates were used for the limit concentration and the control. The environmental conditions were within the acceptable limits.

 

The concentration of the test item N-acetylhexanelactam was analytically verified via HPLC-DAD at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in the limit concentration and the control.

The measured concentration of the test item at the start of the exposure (0 hours) was 100% of the nominal value. The measured concentrations at the end of the exposure (72 hours) was 62% of the nominal value. The geometric mean measured concentration was 78.7 mg/L.

 

All effect values were based on the geometric mean measured test item concentration of the test item N-acetylhexanelactam.In this study, no inhibition of growth on the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test itemN-acetylhexanelactam.The results are summarized in the following table:

 

NOEC, LOEC, EC - valuesofN-acetylhexanelactam(0-72hours)

                  based on the geometric mean measured test concentration [mg/L]

 

	Inhibition of Growth Rate
NOEC	78.7
LOEC	>78.7
ErC10	>78.7
ErC20	>78.7
ErC50	>78.7
	Inhibition of Yield
NOEC	78.7
LOEC	>78.7
EyC10	>78.7
EyC20	>78.7
EyC50	>78.7