3-dodecyl-1-(2,2,6,6-tetramethyl-4-piperidyl)pyrrolidine-2,5-dione

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

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 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). Due to 100% mortality after 24 hours, the nominal concentration level of 62.5% of the saturated solution were not analysed anymore during the second exposure interval (24 and 48 hours).
The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000). Details of the analytical method are presented in section 15. Analytical results are presented in section 8.2.3.

Sampling for the analytical monitoring
At the start of the exposure intervals (0 and 24 hours), sampling was
carried out from the newly prepared test concentrations.
At the end of the exposure intervals (24 and 48 hours), samples were taken directly from the test vessels.

Quality criteria for the analytical monitoring
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
A dispersion with a nominal concentration of 100 mg/L of the test item
was prepared with dilution water (ISO test water as specified below) one day prior to the start of each exposure interval (at -24 and 0 hours). An appropriate amount of the test item was weighed out and transferred with an appropriate amount of dilution water into a measuring flask. This dispersion was stirred for 24 hours with approximately 1100 rpm at room temperature. Then, undissolved test item was removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used as stock solution for preparation of the test concentrations by diluting with ISO test water. During filtration, the filter was always kept covered.

Test concentrations
5 concentration levels of the test item in a geometric series with a separation factor of 2.5, prepared by diluting the saturated solution with dilution water, were tested as follows:
1.60 - 4.00 - 10.0 - 25.0 - 62.5% of the saturated solution.

The concentration rates mentioned above correspond to the geometric mean measured concentrations of:
55.7 - 144 - 419 - 778 - 2971 µg/L.
The concentration rates mentioned above are selected based on the results of a non GLP preliminary range finding test (see section 8.1).

Control
Dilution water without test item tested under the same conditions as the test groups

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. 20 µEm-2  s-1

Culture medium
Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was used. The composition of the culture medium is presented in Table 2.

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

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Dilution water dated:

0 hours: 2015-09-16 Total hardness [mg CaCO3/L]: 274
24 hours: 2015-09-17 Total hardness [mg CaCO3/L]: 271

Test temperature:

18 - 22°C, constant within ± 1°C
2015-09-16 20.6 °C
2015-09-17 20.0°C

pH:

Water Quality Parameters at the End of the Exposure Interval (24 hours)

Geometric mean
measured concentration
of the test item
[µg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
2971 7.92 7.92 7.91 7.91 8.60 8.58 8.56 8.58
778 7.89 7.89 7.90 7.90 8.63 8.61 8.64 8.65
419 7.86 7.85 7.87 7.87 8.64 8.64 8.59 8.62
144 7.85 7.86 7.87 7.88 8.65 8.67 8.66 8.68
55.7 7.83 7.84 7.86 7.85 8.61 8.60 8.63 8.59
Control 7.63 7.75 7.82 7.82 8.50 8.52 8.61 8.59

Dissolved oxygen:

Water Quality Parameters at the End of the Exposure Interval (24 hours)

Geometric mean
measured concentration
of the test item
[µg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
2971 7.92 7.92 7.91 7.91 8.60 8.58 8.56 8.58
778 7.89 7.89 7.90 7.90 8.63 8.61 8.64 8.65
419 7.86 7.85 7.87 7.87 8.64 8.64 8.59 8.62
144 7.85 7.86 7.87 7.88 8.65 8.67 8.66 8.68
55.7 7.83 7.84 7.86 7.85 8.61 8.60 8.63 8.59
Control 7.63 7.75 7.82 7.82 8.50 8.52 8.61 8.59

Nominal and measured concentrations:

Method of determination
Analytical evaluation of test item was carried out via LC-MS/MS on a reversed-phase column in gradient mode. An electrospray tandem mass spectrometer operating in positive ion mode was used as detector. An external standard calibration was used.

Equipment Autosampler: Acquity UPLC, WATERS
Binary Solvent Manager: Acquity UPLC, WATERS
Column Manager: Acquity UPLC, WATERS
Detector: Mass selective detector, Xevo TQD MS, Acquity UPLC, WATERS
Software: MassLynxTM 4.1, WATERS

Additional equipment
Piston stroke pipettes, THERMO SCIENTIFIC
Direct displacement pipettes, GILSON MEDICAL

Reagents HPLC water, for HPLC, VWR
Acetonitrile, super gradient, VWR
Formic Acid, ≥ 98%, SIGMA-ALDRICH


Analytical Standard
The test item was used for the preparation of the external standards.

Details on test conditions:

Test method
The study was performed with a semi-static test design (with a renewal of the test solutions after 24 hours), since the concentration of the test item in the preliminary test decreased markedly within 48 hours

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Renewal of the test solutions T
he test solutions were renewed after 24 hours. For this purpose, a second set of test vessels were filled with the freshly prepared test solutions and the
daphnids were transferred by pipette (see ‘Application’).


Dilution water
ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.75
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123

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

Age of the daphnids at the start of the exposure
2 to 24 hours old daphnids from a healthy stock were used for the
study. Juvenile daphnids were removed from the culture vessels latest 22 hours before the start of the exposure and discarded. The juveniles born within this period of max. 22 hours preceding the exposure were used for the test after an acclimatisation phase of at least 2 hours in the dilution water. No first brood progeny was used for the test.

Acclimatisation
At least 2 hours in dilution water

Application
20 g test solution per replicate were weighed out into the test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette.

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

Illumination (target)
Diffuse light, light intensity of max. 20 µEm-2  s-1 (max. 1340 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

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

517 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

333 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

808 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

501 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

144 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 419 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

2 971 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

2 971 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Results with reference substance (positive control):

A reference test was conducted as an acute immobilisation test (acc. to AQS P 9/2 and OECD 202) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 8.3.

Reference item Potassium dichromate p.a. (SIGMA)

Purity 100.0%

Batch No. MKBQ9179V

Storage stability 2015-12-29

Test concentrations 1.00 – 2.00 – 4.00 mg/L

Ranges of validity 0.6 mg/L < EC50 (24 h) < 2.4 mg/L, according to AQS P 9/2 (clone 5),
0.6 mg/L < EC50 (24 h) < 2.1 mg/L, according to OECD 202 (clone A)

Test duration 2015-09-02 to 2015-09-03




The percentage immobility for the reference item was determined after 24 hours. The EC50-value with 95% confidence limits (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.92 mg/L (CI 1.85 - 1.99 mg/L)
The EC50-value of the reference item potassium dichromate after 24 hours is within the required concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202

Reported statistics and error estimates:

Methods of evaluation
The EC100-values (after 24 and 48 hours), the NOEC and LOEC (after 48 hours) were deduced directly from the observation data.
Since the measured concentrations did not remain within ± 20% of initially measured concentrations, the effect levels (EC10 / 50 / 100, NOEC and LOEC) were based on the geometric mean measured concentrations the test item Hostavin 3055.

EC-values and statistical analyses
EC10- and EC50-values after 24 and 48 hours were calculated by
sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5. The concentration effect relationships are shown graphically. The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.

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

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item, the 48 hour EC50 for Daphnia magna was 501 µg/L (95% confidence limits: 460 – 589 µg/L).
The NOEC after 48 hours was 144 µg/L. The LOEC after 48 hours was 419 µg/L.

Executive summary:

    Summary

 

In the acute immobilisation test with Daphnia magna(STRAUS), the effects of the test item were determined according to OECD 202 (2004) from2015-09-15 to 2015-09-18, with the definitive exposure phase from 2015-09-16 to 2015-09-18,at the test facility.

The study was conducted under semi-static conditions over a period of 48 hours with five dilution levels of a saturated solution (prepared with a nominal concentration of100 mg/L of the test item) in a geometric series with a separation factor of 2.5 (nominal concentrations of 1.60 to 62.5%).

The test item is a clear, medium viscous, slightly yellowish liquid with a water solubility of < 0.01 g/L (22 °C).A saturated solution with a nominal concentration of 100 mg/L of the test itemwas prepared with dilution water (ISO test water, acc. to OECD 202, Annex 3) one day before the start of the exposure intervals (at -24 and 0 hours) and was used as stock solution for the preparation of all tested dilution levels.

A dispersion with a nominal concentration of 100 mg/L prepared in ISO test water was stirred with approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, the stock solution was filtered with a membrane filter (0.45 µm, RC, Macherey-Nagel) in order to remove undissolved test item. The filter was saturated with dissolved test item in order to avoid adsorption during the filtration.

The tested dilutions of the saturated solution were visually clear throughout the exposure period.

Twenty daphnids were exposed to each concentration leveland the control.

The concentrations of the test item Hostavin 3055wereanalytically verified via LC-MS/MS at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all tested concentration levels, the stock solution and the control. Details of the analytical method are presented.

The measured concentrations of the saturated solution were 5215 µg/L at 0 hours and 4612 µg/L at 24 hours. The measured concentrations of the test item at the start of the exposure intervals were within the range of 89.9 to 3239 µg/L at 0 hours for the nominal concentrations of 1.60 to 62.5% of the saturation and 63.8 to 639 µg/L at 24 hours for the nominal concentrations of 1.60 to 25.0% of the saturation. Due to 100% mortality after 24 hours, the nominal concentration level of 62.5% was not analysed anymore during the second exposure interval.
At the end of the exposure intervals (24 and 48 hours), the measured concentrations were within the range of 45 to 110% of the initially measured concentrations.The analytical results arepresented.

Since the measured test item concentrations did not remain within±20% of the initially measured concentrations, the effect values given are based on the geometric mean measured concentrations of the test item. The geometric mean measured concentrations were calculated to be: 55.7 - 144 - 419 - 778 - 2971 µg/L.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours), were within the acceptable limits. The validity criteria of the test guidelines were fulfilled.

 

 

   EC₁₀-, EC₅₀- (with 95% Confidence Limits), EC₁₀₀-Values, NOEC and LOEC

(based on the geometric mean measured concentrations of the test item)

Test item
Effect levels	Test duration   [hours]	Toxicity endpoint based on the geometric mean measured concentrations the test item [µg/L]
EC10 (with 95% confidence limits)	24	517	(Cl: 342 – 692)
	48	333	(Cl: < 55.7 – 390)
EC50 (with 95% confidence limits)	24	808	(CI: 649 – 1167)
	48	501	(Cl: 460 – 589)
EC100	24	2971
	48	2971
NOEC	48	144
LOEC	48	419

 

Description of key information

OECD 202 (Daphnia magna; semistatic; mean measured; RL 1):

EC10 (48 h; immobilisation) = 333 µg/L (95% CI: <55.7 - 390);

EC50 (48 h; immobilisation) = 501 µg/L (95% CI: 460 - 589)

EC100 (48 h; immobilisation) = 2971 µg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

501 µg/L

Additional information

An acute toxicity test on Daphnia magna (semi-static, renewal after 24 hours) was performed with the submission substance according to OECD Guideline 202 compliant with GLP (RL1; Noack, 2016). All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). The following 5 concentration levels of the test item were applied, given in % of the saturated solution (diluted with test water): 1.60 - 4.00 - 10.0 - 25.0 - 62.5%. These concentration levels corresponded to the following geometric mean measured concentrations: 55.7 - 144 - 419 - 778 - 2971 µg/L. 20 daphnids divided into 4 replicates were used for each concentration level and the control.

The following effect concentrations (based on geometric mean measured values) had been determined:

EC10 (48 h; immobilisation) = 333 µg/L (95% CI: <55.7 - 390);

EC50 (48 h; immobilisation) = 501 µg/L (95% CI: 460 - 589)

EC100 (48 h; immobilisation) = 2971 µg/L