1-acetyl-4-(3-dodecyl-2,5-dioxo-1-pyrrolidinyl)-2,2,6,6-tetramethylpiperidine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-11-08 to 2000-11-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentration were sampled at the beginning and before each media renewal (renewal of test media on day 0, 2, 5, 7, 9, 12, 14, 16, 19, and 21)
- Sample storage conditions before analysis: The samples were analysed immediately and without pre-treatment.

Vehicle:

yes

Details on test solutions:

PREPARATION OF TEST SOLUTIONS
For the purpose of the definitive study the test material was prepared by using a preliminary solution in dimethylformamide. Amounts of test material (100 and 32 mg) were each separately dissolved in dimethylformamide and the volume adjusted to 100 ml to give the 100 and 32 mg/100 ml solvent stock solutions. Serial dilutions were made from these to give further solvent stock solutions of 1.0, 0.32, 0.10 and 0.032 mg/10 ml. Aliquots (200 μI) of the 0.032, 0.10, 0.32 and 1.0 mg/10 ml and 32 mg/100 ml solvent stock solutions were each separately dispersed in reconstituted water and the volume adjusted to 2 liters to give the 0.00032, 0.0010, 0.0032, 0.010 and 0.032 mg/l test concentrations, respectively. Each of the solvent stock solutions and prepared test concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test solutions were verified by chemical analysis on days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 (old and fresh media) and 21 (old media).

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamid (100 µg/L)
- Dimethylformamid concentration in the solvent control: 100 µg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): yes (see also analytical method)

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Name: Daphnia magna
- Source: in-house laboratory cultures
- Feeding during test: yes
- Food type: unicellular algal culture (Chlorella sp.)
- Amount: approximately 0.1 mg carbon/daphnid
- Frequency: daily

PARENTAL STOCK AND METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 liters of reconstituted water at a temperature of 21°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods at a light intensity preferably not exceeding 600 lux. Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced over night were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

241 - 270 mg/L CaCO3

Test temperature:

21 °C

pH:

Control: 7.9 - 8.0
Solvent control: 7.9 - 8.0
Treatment groups: 7.9 - 8.0

Dissolved oxygen:

Control: 8.0 - 8.4 mg O2/L
Solvent control: 8.0 - 8.4 mg O2/L
Treatment groups: 8.0 - 8.4 mg O2/L

Nominal and measured concentrations:

Nominal concentrations: 0.00032, 0.0010, 0.0032, 0.010 and 0.032 mg/L
Mean measured concentrations (TWA):

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 ml glass flasks, covered with plastic lid
- Fill volume: 100 mL
- Aeration: no; diluent water was aerated prior to use
- Renewal rate of test solution (frequency): The test solutions were renewed 3 times per week on days 2, 5, 7, 9, 12, 14, 16, and 19.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS / MEASUREMENT INTERVALS
The reconstituted water used for the range-finding and definitive studies was the same as that used to maintain the stock animals. For preparation of the reconstituted water see Any other information on materials and methods .

Temperature of the test solutions and light intensity were recorded daily throughout the study. Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. Measurements were made on one replicate for each test concentration. The water hardness of the solvent control and the highest surviving test concentration in the fresh and old media was measured at least once per week.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light
- Light intensity: 106 - 126 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- number of live and dead offspring, daily
- number of discarded unhatched eggs, daily
- immobilisation of parental generation, daily
- general condition and size of the parental daphnids, daily
- length of each surviving parent animal, end of the study

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.0010, 0.010, 0.10 and 0.80 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

(parental)

Remarks on result:

other: 95% CL: 0.0033 - 0.0096 mg/L

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.003 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ immobilisation (parental)

Remarks on result:

other: 95% CL: 0.0023 - 0.0042 mg/L

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ immobilisation (parental)

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

0.018 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

(parental)

Remarks on result:

other: 95% CL: 0.010 - 0.032 mg/L

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.009 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ immobilisation (parental)

Remarks on result:

other: 95% CL: 0.0063 - 0.013 mg/L

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.003 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

/ immobilisation (parental)

Details on results:

RECOREDED RESULTS AND OBSERVATIONS

For result data tables see Any other information on results.

Mortality (immobilisation) occurred at the highest test concentration of nominal 0.032 mg/l resulting in 80% and 100% mortality by days 7 and 9, respectively. Significant mortality (immobilisation) also occurred in the latter stages of the study in the nominal 0.010 mg/l test group resulting in 20% and 60% mortality by days 16 and 21, respectively, indicating a prolonged toxic effect attributable to exposure of Daphnia magna to the test material. No mortalities occurred at nominal 0.00032, 0.0010 and 0.0032 mg/l test concentrations throughout the study.
Information on the effects of the test material on the filial generation was limited because by study design the young daphnids were removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the young daphnids produced by nominal 0.00032, 0.0010, 0.0032 and 0.010 mg/l test groups were in the same general condition as the young produced by the solvent controls over the duration of the study. Young daphnids were first produced in the solvent control test group on day 7 of the study. All test item concentrations with exception of the highest (death of parental animals before the onset of reproduction) had this same time to production of first brood. Due to the toxic effect of the test material the parental generation of the 0.032 mg/l test group were eliminated prior to the production of young. Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

Preliminary chemical analysis showed that the test material was removed from the test diluent by filtration (0.2 µm) and centrifugation, indicating it to be dispersed throughout the diluent rather than being in solution. This work also showed that the test material adsorbed to algal cells, which are used as feed during the study.

OBSERVATION ON TEST MATERIAL SOLUBILITY
Throughout the study the freshly prepared test media were observed to be clear, colourless solutions, whereas the old test media were observed to be green tinged solutions due to the presence of algae used as feed for the daphnids.

VALIDATION CRITERIA
The following validation criteria were achieved during the study:
- Solvent control mortality: 0% (required <=20%)
- Dissolved oxygen: >= 8.0 mg O2/L (required >3 mg O2/L)
- pH (solvent control group): deviation 0.1 (required <= 1.5)
- Mean number of live young per surviving adult (solvent control group): 89 (required >=60 after 21 days)
- Coefficient of variation for solvent control group: 19% (required <=25%)

Reported statistics and error estimates:

STATISTICS
The EC50 (immobilisation; reproduction) values and associated confidence limits at days 5, 7 and 21 were calculated by the trimmed Spearman-Karber method of Hamilton et al. (1977)* using the ToxCalc computer software package (Version 5.0.23C) and at day 14 by the geometric mean method.
When only one partial response was shown the trimmed Spearman-Karber method was appropriate. If there was no immobilisation between 0% and 100% immobilisation, then the geometric mean of the highest test concentration showing no immobilisation and the lowest test concentration showing 100% immobilisation was calculated. The concentrations resulting in 0% and 100% immobilisation were the 95% confidence limits.
The NOEC was calculated using one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control. All statistical analyses were performed using the SAS computer software package (Release 6.12).
The 0.010 mg/l and 0.032 mg/l test group data was not included in the statistical analysis due to the observation of statistically significant mortalities in the parental (P1) generations.

*) Hamilton, M A, Rtrso R C and Thurston, R V (1977) Trimmed Spearman-Karber, Method for Estimating Median Letal Concentration in Toxicity Bioassays. Environ Sci Technol 11 (7):714-719.

Table 1                Summary of Findings Following the Exposure of Daphnia magnafor 21 days

		Number of Live Young*		Number of Dead Young*		Number of Unhatched Eggs*
Nominal Concentration (mg/l)	% Survival of P1	Total	Per Female (cumulative)	Total	Per Female (cumulative)	Total	Per Female (cumulative)
Control	100	871	87	1	<1	4	<1
Solvent Control	100	890	89	1	<1	2	<1
0.00032	100	877	88	2	<1	0	0
0.0010	100	865	87	0	0	1	<1
0.0032	100	879	88	2	<1	3	<1
0.010	40	335	34	0	0	0	0
0.032	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Remarks:

according to OECD 211 (adopted 1998; 2012)

Conclusions:

The following effect concentrations on the reproductive success and parental immobilisation of Daphnia magna were obtained (based on measured concentrations (TWA)): the 21d-EC50 = 0.0031 mg/l and the 21d-NOEC = 0.0013 mg/l.

Executive summary:

A study was performed to assess the effect of the test material on the reproduction of Daphnia magna over a 21 days period. The method followed the OECD Guidelines for Testing of Chemicals No 211 (1998) and was in compliance with GLP.

Based on the results of a preliminary range finding study, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous dispersion of the test material over a range of nominal test concentrations of 0.00032, 0.0010, 0.0032, 0.010 and 0.032 mg/l for a period of 21 days. The test dispersions were renewed 3 times per week. The number of live as well as dead adult Daphnia, and young daphnids (live and dead) were determined daily.

Analysis of the fresh test preparations showed measured test concentrations to range from 84% to 118% of nominal values, indicating that the test system was correctly dosed. However, a marked decline was shown from analysis of the expired test media with all measured values being less than the limit of quantitation (LOQ) with the exception of the 0.010 mg/l test group on day 2 and the 0.032 mg/l test group on day 5, which showed measured test concentrations of 22% and 17%, respectively. Analysis was not performed for the 0.00032 mg/l test group from day 9 onwards, as this test concentration was shown to be less than the LOQ (LOQ < 0.00080 mg/l).

The marked decline in the measured test concentrations shown over each renewal period was considered to be due to adsorption of the test material to the algal cells used as food for the daphnids and possible ingestion of the test material directly by the filter feeding actions of the test organisms. Given the marked decline in measured test concentrations the results were based on the time-weighted mean measured concentrations (TWA).

Exposure of Daphnia magna to the test material resulted in a significant prolonged mortality effect at the highest test concentration of 0.0096 mg/L resulting in 100% mortality by day 9. Significant mortality was also observed at 0.0033 mg/L test concentration resulting in 20% mortality by day 16 and 60% mortality on day 21. The following effect concentrations were determined: the 21d-EC50 (reproduction, parental immobilisation) = 0.0031 mg/l and the 21d-NOEC (reproduction, parental immobilisation = 0.0013 mg/l.

The study fulfilled the validity criteria of the OECD guideline 2011 (1998; 2012) and results were considered reliable.

Description of key information

21d-EC50 (Daphnia magna, reproduction/parental immobilisation) = 0.0031 mg/l (TWA); 21d-NOEC (Daphnia magna, reproduction/parental immobilisation) = 0.0013 mg/l

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.001 mg/L

Additional information

A reliable study was performed to assess the effect of the test material on the reproduction of Daphnia magna over a 21 days period. The method followed the OECD 211 (1998) and was in compliance with GLP (RL1).

Based on the results of a preliminary range finding study, Daphnia magna were exposed to an aqueous dispersion of the test material over a range of nominal test concentrations of 0.00032, 0.0010, 0.0032, 0.010 and 0.032 mg/l under semi-static test conditions.

The marked decline in the measured test concentrations shown over each renewal period was considered to be due to adsorption of the test material to the algal cells used as food for the daphnids and possible ingestion of the test material directly by the filter feeding actions of the test organisms. Given the marked decline in measured test concentration, determined effect concentrations were based on the time-weighted mean measured concentrations (TWA).

Exposure of Daphnia magna to the test material resulted in a significant prolonged mortality effect at the highest test concentration of 0.0096 mg/L resulting in 100% mortality by day 9. Significant mortality was also observed at 0.0033 mg/L test concentration resulting in 20% mortality by day 16 and 60% mortality on day 21. The following effect concentrations were determined: the 21d-EC50 (reproduction, parental immobilisation) = 0.0031 mg/l (TWA), and the 21d-NOEC (reproduction, parental immobilisation) = 0.0013 mg/l (TWA).

The study fulfilled the validity criteria of the OECD guideline 2011 (1998; 2012), and results were considered reliable.