N-acetylhexanelactam

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-12-06 to 2017-12-08

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation 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:

Determination of the test item
The limit concentration and the control were analytically verified via HPLC-DAD in fresh media at the start of exposure and at water renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the exposure (24 and 48 hours). The samples were analysed with a HPLC-DAD method, implemented under non-GLP and documented finally in the GLP raw data.

Sampling for the analytical monitoring
At the start of the exposure and at renewal (0 and 24 hours), samples of the freshly prepared test item limit concentration were taken and analyzed.
At renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels and analyzed.

Criteria for the analytical monitoring (target)
Recoveries of the active substance should be within ± 20% of the nominal or initially measured concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

Limit concentration
A limit concentration of 100 mg/L of the test item N-acetylhexanelactam was tested. The limit concentration is based on the results of a non-GLP preliminary range finding test.

Preparation of the limit concentration The limit concentration (100 mg/L of the test item was weighed out) was freshly prepared with dilution water (see Table 2 and adjusted to pH 6.0 – 6.2 by addition of 1 M HC) prior to the start of the exposure (at 0 hours) and the renewal of the test solutions (at 24 hours). The limit concentration was mixed thoroughly by manual agitation.

Control
Dilution water (adjusted to pH 6.0 – 6.2 by addition of 1 M HCl) without test item incubated under the same conditions as the test groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5).

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2°C, in an incubator, 16 hours illumination, light intensity of max. 1500 lx

Culture medium
Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was used.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany


Composition of the Culture Medium according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2  0.8

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 172
24 hours:Total hardness [mg CaCO3/L]: 174

Test temperature:

18 - 22°C, constant within ± 1°C
20.6 - 20.7 °C

pH:

see any other information on materials and methods incl. tables

Dissolved oxygen:

see any other information on materials and methods incl. tables

Conductivity:

see any other information on materials and methods incl. tables

Details on test conditions:

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids were
used for each concentration level and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for
the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization was not necessary, because the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by pipette.

Test temperature (target)
18 - 22°C, constant within ± 1°C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

other: EC10/50/100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EC10/50/100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017-11-02 to 2017-11-03. For results of the most recent of the monthly performed reference tests, see the table below.

EC50-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.64 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 1.00 - 4.00 mg/L

Reported statistics and error estimates:

Methods of evaluation
The effects of the limit concentration were deduced directly from the observed immobilization rates.
Since the measured concentrations were within ± 20% of the nominal value, the effect levels (EC10 / 50 / 100) were based on the nominal concentration of the test item.

ECx-values and statistical amalyses and for reference item An EC50-value was calculated for the reference item by linear dose-response regression. The respective 95 % confidence limits for the EC50 were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

                (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	0	0	0	0	0	20	0	0	0	5
Control	0	0	0	0	0	0	0	0	0	0

 

 Absolute Numbers of immobile Daphnids after 24 and 48 hours of Exposure in the Definitive Test

                (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
100	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	1 / 5	0 / 5	0 / 5	0 / 5	1 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the limit concentration 100 mg/L of the test item N-acetylhexanelactam, no effects on Daphnia magna were observed.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item N-acetylhexanelactam were determined at the test facility according to OECD 202 (2004) from 2017-12-06 to 2017-12-08.

The study was conducted under semi-static conditions over a period of 48 hours with the limit concentration of 100 mg/L of the test item N-acetylhexanelactam.The limit concentration was mixed thoroughly by manual agitation until it was completely dissolved.The limit concentration was visually clear throughout the exposure.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to the limit concentration and the control.

The concentration of the test item N-acetylhexanelactam was analytically verified via HPLC-DAD in fresh media of the limit concentration and in the control at the start of the exposure and at renewal (0 and 24 hours) and in 24-hours old media at renewal and at the end of the test (24 and 48 hours).

The measured concentration of the test item in fresh media at the start of the exposure and at renewal (0 and 24 hours) was 102% of the nominal value and 96% and 100% in old media at renewal and at the end of the test (24 and 48 hours).

The measured test item concentration was within ± 20% of the nominal concentrations. This indicates that the test item concentration was successfully maintained for the duration of the test.

Since the measured test item concentration was within the range of ± 20% of the nominal concentrations, the effect concentrations given in the table below are based on nominal concentrations of the test item N-acetylhexanelactam.

The validity criteria of the test guidelines were fulfilled.

 

 

In the limit concentration100 mg/Lof the test item N-acetylhexanelactam,

no effects on Daphnia magna were observed.

 

 

 

EC₁₀-, EC₅₀-, EC₁₀₀-Values

(based on the nominal concentration of the test item)

Effect concentrations	Test duration [hours]	Toxicity endpoint based on the nominal concentration the test item [mg/L]
EC10 / 50 / 100	24	> 100
	48	> 100