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

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:

2016-02-01 to 2016-02-19

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
Analytical evaluation of the concentrations of the test item was carried out via LC-MS/MS from freshly prepared media after 0 hours and old test media after 72 hours of exposure. Samples for the test item analysis at the beginning of the exposure were taken from additionally prepared replicates with algae. For the test item analysis after 72 hours, samples were taken from the test replicates.

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
A dispersion with a nominal loading of 100 mg/L of the test item was prepared with dilution water one day prior to the start of the exposure. 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 was discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL was discarded. The following filtrate, i.e. the saturated solution, was used as highest loading level in the test and for preparation of further loading rates by diluting with dilution water. During filtration, the filter was always kept covered.

Test loadings
5 loading rates 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:
1.02 - 2.56 - 6.40 - 16.0 - 40.0 - 100 % of the saturated solution.
The loading levels are based on the results of a preliminary range finding test (non GLP)

Test concentrations (geometric mean measured)
19.7 – 51.0 – 129 – 330 – 869 – 2299 µg/L


Control
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): 4 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:

not specified

Test temperature:

min.: 21.0 max.: 22.0, mean value: 21.5 °C

pH:

Geometric mean measured test item concentration pH-value
[µg/L] Start; 0 hours End; 72 hours
2299 7.39 7.38
869 7.47 7.40
330 7.54 7.55
129 7.64 7.66
51.0 7.73 7.78
19.7 7.85 7.92
Control 7.90 8.13

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

See window "Any other information on materials and methods incl. tables"

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 4847 cells/mL
- Control end cells density: Mean 1358509 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: 60 - 120 µE x m^-2 x s^-1, mean value: 70.6 µE x m^-2 x s^-1,



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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

129 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

330 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.:

263 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 186 - 311

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

300 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 220 - 322

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

374 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 344 - 561

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

129 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

330 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

134 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 116 - 160

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

163 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 143 - 187

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

229 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 207 - 250

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):

The toxicity of potassium dichromate (SIGMA, batch number MKBQ9179V, purity 100.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 hours from 2015-10-19 to 2015-10-22. 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 0.685 0.671 ± 0.401
95 % confidence interval 0.603 - 0.794
Yield Inhibition
EyC50 0.362 0.320 ± 0.139
95 % confidence interval 0.328 - 0.398
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses

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

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:
Growth rate and yield: Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01.
Growth rate and yield: Levene’s test on variance homogeneity was done with a significance level 0.01.
Growth rate and yield: Monotonicity of concentration/response was done by trend analysis by contrasts (significance level 0.05).
Growth rate: Step-down Jonckheere-Terpstra’s test on significant differences was done with a significance level 0.05.
Yield: Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm was done with a significance level 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

Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[µg/L]	No.	0 hours	24 hours	48 hours	72 hours
2299	1	4847	10658	684*	684*
	2	4847	10802	1763	684*
	3	4847	13260	1768	684*
	Mean	4847	11573	1405	684*
869	1	4847	20110	6107	684*
	2	4847	22323	4111	4313
	3	4847	21073	7016	2081
	Mean	4847	21169	5745	2359
330	1	4847	25017	37149	137665
	2	4847	30063	46756	237535
	3	4847	29005	75032	386295
	Mean	4847	28028	52979	253832
129	1	4847	39747	235510	1172711
	2	4847	35442	254396	1288837
	3	4847	36841	237704	1257159
	Mean	4847	37343	242537	1239569
51.0	1	4847	43076	305522	1352239
	2	4847	39155	338955	1449315
	3	4847	43744	310814	1379899
	Mean	4847	41992	318430	1393818
19.7	1	4847	40921	323081	1374175
	2	4847	42417	291812	1320490
	3	4847	39699	312497	1294321
	Mean	4847	41012	309130	1329662
Control	1	4847	34576	297248	1276377
	2	4847	34672	288955	1503625
	3	4847	36211	292899	1653617
	4	4847	38482	317279	1264592
	5	4847	44793	254310	1238326
	6	4847	30934	291225	1214514
	Mean	4847	36611	290319	1358509

 

*) values below the limit of quantification (1368 cells/mL) are given as ½ limit of quantification

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		Inhibition of growth rate	Yield		Inhibition of yield
[µg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
2299	1		-0.653*	100		-4163*	100
	2		-0.653*	100		-4163*	100
	3		-0.653*	100		-4163*	100
	Mean	(+)	-0.653*	100	(+)	-4163*	100
869	1		-0.653*	100		-4163*	100
	2		-0,039*	100		-534*	100
	3		-0,282*	100		-2766*	100
	Mean	(+)	-0.324*	100	(+)	-2487*	100
330	1		1.12	41		132818	90
	2		1.30	31		232688	83
	3		1.46	22		381448	72
	Mean	(+)	1.29	31	(+)	248985	82
129	1		1.83	2		1167864	14
	2		1.86	1		1283990	5
	3		1.85	1		1252312	7
	Mean	(-)	1.85	2	(-)	1234722	9
51.0	1		1.88	0		1347392	0
	2		1.90	-1		1444468	-7
	3		1.88	0		1375052	-2
	Mean	(-)	1.89	-1	(-)	1388971	-3
19.7	1		1.88	0		1369328	-1
	2		1.87	0		1315643	3
	3		1.86	1		1289474	5
	Mean	(-)	1.87	0	(-)	1324815	2
Control	1		1.86			1271530
	2		1.91			1498778
	3		1.94			1648770
	4		1.86			1259745
	5		1.85			1233479
	6		1.84			1209667
	Mean		1.88			1353662

Negative values = growth stimulation

 

*) calculated values, considered to be 0.

 

 

Validity criteria fulfilled:

yes

Conclusions:

In this study, the substance was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values (based on geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 374 (344 – 561) and 229 (207 – 250) µg/L. The NOEC-values for inhibition of growth rate and yield after 72 hours were for both 129 µg/L.

Executive summary:

Summary

 

The toxicity of the substance to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No. 761/2009/Method C.3 (2009) at the test facility from 2016-02-01 to 2016-02-19 with the definitive exposure phase from 2016-02-16 to 2016-02-19.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 4847 cells/mL. Based on a preliminary range finding test a saturated solution with a nominal loading of 100 mg test item/L was prepared in dilution water. The dispersion was stirred for 24 hours with approximately 1100 rpm at room temperature. At the end of the stirring period, undissolved test item was removed by membrane filtration (membrane filter 0.45 µm, RC, Macherey-Nagel). The saturated solution and further five dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2.5: 1.02 - 2.56 - 6.40 - 16.0 - 40.0 - 100 % of the saturated solution corresponding to geometric mean measured concentration levels of: 19.7 – 51.0 – 129 – 330 – 869 – 2299 µg/L.Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

 

All test concentrations of the test item and the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 hours) and the end of the exposure (72 hours).
The measured concentrations of the test item at the start of the exposure were in the range of 21.8 to 2564 µg/L. At the end of the exposure, they were in the range of 17.8 to 2062 µg/L.
All effect values given are based on the geometric mean measured test item concentrations.

NOEC, LOEC, EC - values of the test item (0-72 hours)

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

                               

	Growth Rate Inhibition geometric mean measured test item concentration [µg/L]
NOEC	129
LOEC	330
ErC10	263 (186 – 311)
ErC20	300 (220 – 322)
ErC50	374 (344 – 561)
	Inhibition of Yield geometric mean measured test item concentration [µg/L]
NOEC	129
LOEC	330
EyC10	134 (116 – 160)
EyC20	163 (143 – 187)
EyC50	229 (207 – 250)

 

 

 

    

                               

 

Description of key information

OECD 201 (Desmodesmus subspicatus; static; mean measured; RL 1):

NOEC (72 h; growth rate): 129 µg/L;

LOEC (72 h; growth rate): 330 µg/L;

EC10 (72 h; growth rate): 263 µg/L (95% CI: 186 - 311);

EC50 (72 h; growth rate): 374 µg/L (95% CI: 344 - 561).

Key value for chemical safety assessment

EC50 for freshwater algae:

374 µg/L

EC10 or NOEC for freshwater algae:

263 µg/L

Additional information

The toxicity of the substance to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No. 761/2009/Method C.3 (2009) compliant with GLP (RL1; Noack, 2016).

The study was conducted under static conditions with an initial cell density of 4847 cells/mL. Based on a preliminary range finding test a saturated solution with a nominal loading of 100 mg test item/L was prepared in dilution water. After stirring for 24 hours at room temperature undissolved test item was removed by membrane filtration. The following 6 concentration levels of the test item were applied, given in % of the saturated solution (diluted with test water): 1.02 - 2.56 - 6.40 - 16.0 - 40.0 - 100 %. These concentration levels corresponded to the following geometric mean measured concentrations: 19.7 – 51.0 – 129 – 330 – 869 – 2299 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

All test concentrations of the test item and the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 hours) and the end of the exposure (72 hours). The measured concentrations of the test item at the start of the exposure were in the range of 21.8 to 2564 µg/L. At the end of the exposure, they were in the range of 17.8 to 2062 µg/L.  pH values within treatment groups were between 7.38 and 7.92, pH for the controls was between 7.9 and 8.13.

All effect values given are based on the geometric mean measured test item concentrations:

NOEC (72 h; growth rate): 129 µg/L;

LOEC (72 h; growth rate): 330 µg/L;

EC10 (72 h; growth rate): 263 µg/L (95% CI: 186 - 311);

EC50 (72 h; growth rate): 374 µg/L (95% CI: 344 - 561).