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

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:

2000-10-31 to 2000-11-03

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 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(92/69/EEC, C.3)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

SAMPLING
Sampling was always in duplicate for each determination of concentration, as one sample was analyzed without pre-treatment, while the other was analyzed after centrifugation at 17 000 G for 30 min. At the start (0 hours) and after 72 hours (end of the test) the following test groups were analyzed: solvent control, pooled treatment replicates 1-3 (R1-R3), pooled treatment replicates 4-6 (R4-R6). All treatment replicates (R1-R6) were of the same concentration (limit test).

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
An amount of test material (200 mg) was dissolved in dimethylformamide and the volume adjusted to 25 ml to give a 200 mg/25 ml solvent stock solution. An aliquot (300 µI) of this solvent stock solution was dispersed in 3 litres of algal suspension to give the required test concentration of 0.80 mg/l. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test material in the centrifuged and uncentrifuged test samples were verified by chemical analysis at the start of the test and after 72 hours (see Any other information on materials and methods).

During preliminary solubility work precipitation of the test material was observed (by visual inspection) at concentrations in excess of 0.80 mg/l indicating this to be the maximum limit of water solubility under these test conditions. Based on this information a single test concentration of six replicates of 0.80 mg/l was selected for the definitive study. This experimental design conforms to a limit test to confirm that at the highest attainable test concentration of 0.80 mg/l has no effect on growth.

The test concentration of 0.80 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline. Other various recognised auxiliary solvents were used during preliminary solubility work, however, dimethylformamide was found to give the best testable dispersion of the test material in water. At higher test concentrations there was a marked precipitation of the test material.

It was therefore considered inappropriate to test at concentrations in excess of 0.80 mg/l given that a significant proportion of the test material would not have been in solution and therefore not bioavailable to the algae.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamid (100 µg/L)
- Dimethylformamid concentration in the solvent control: 100 µg/L

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Cumbria. Cultures were maintained in the laboratory by the periodic replenishment of culture medium (see Any other information on materials and methods). The culture was maintained in the laboratory at a temperature of 21 +/- 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

The culture medium used for both the range-finding and definitive studies was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

acc. to culture medium

Test temperature:

24 +/- 1 °C

pH:

Treatment group: (start) 7.3, (end) 9.7
Control: (start) 7.5, (end) 9.8
Solvent control: (start) 7.4, (end) 10.0

Nominal and measured concentrations:

Nominal test concentration: 0.80 mg/L
Measured test concentration: (time-weighted mean): 0.011 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flask
- Fill volume: 100 ml
- Incubation: continuous shaking at approximately 100 rpm for 72 hours
- Initial cells density (nominal) 10^4 cells/ml

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.52 x 10^6 cells per ml. This suspension was diluted to a cell density of 1.40 x 10^4 cells per ml prior to use within the study.

- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: acc. to guideline
- Source/preparation of dilution water: reverse osmosis purified deionised water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured at start of the study and after 72 hours; the temperature was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (Coulter® Multisizer II Particle Counter)
- Counting after 0, 24, 48 and 72 hours

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations (nominal): 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:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.011 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.011 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.011 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.011 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

RESULTS FOR GROWTH RATE AND BIOMASS
Neither the growth (r) nor the biomass (b) of Scenedesmus subspicatus were affected by the presence of the test material over the 72-hour exposure period. There were no statistically significant differences (P >= 0.05) between the solvent control and 0.80 mg/l test group and therefore the NOEC was 0.80 mg/l nominal concentration.

Obtained data showed that the cell concentration of the control cultures increased by a factor of 83 and the cell concentration of the solvent control cultures increased by a factor of 77 during the test. The latter is in line with the OECD guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

For the recorded data see Any other information on results.

OBSERVATIONS
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

TEST CONCENTRATIONS
In order to confirm the correct dosing for the definite test, a sample was prepared in culture medium alone (no algal cells) at a concentration of 0.80 mg/l. Chemical analyses of this sample showed a measured test concentration of 116% of nominal indicating that the correct dosing procedures were followed. Chemical analysis of the test solutions (containing the algea) at 0 hours showed measured concentrations ranging from 50% to 56% of nominal for the uncentrifuged samples and a value of 3% of nominal for the centrifuged samples. This was in line with the recovery analyses which indicated that the test material adsorbed to algal cells. A marked decline was shown in the measured test concentrations at 72 hours ranging from 23% to 24% of nominal for the uncentrifuged samples to less than 1% of nominal for the centrifuged samples. This decline was considered to be due to further adsorption of the test material to algal cells.

It was considered justifiable to base the results on the time-weighted mean measured test concentrations of the centrifuged samples.

PH MEASUREMENTS
The pH values of the control cultures (see any other information on results) were observed to increase from pH 7.5 at 0 hours to pH 9.6 – 9.8 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the guideline (1 pH unit). However, the increase of pH was considered to have had no adverse effect on the results of the study, given that the increase in cell concentration in the control cultures exceeded the validation criterion by far.

Reported statistics and error estimates:

STATISTICAL ANALYSIS
A Students t-test, incorporating Barden' s test for homogeneity of variance, was carried out on the area under the growth curve data at 72 hours for the solvent control and the 0.80 mg/l test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package.

Table 2                Cell Densities and pH Values in the Definitive Study

Nominal Concentration (mg/l)	pH	Cell Densities* (cells per ml)				pH
	0 h	0 h	24 h	48 h	72 h	72 h
Control              R1                         R2                         R3                           Mean	7.5 7.5 7.5	1.12 x 10^4 1.14 x 10^4 1.01 x 10^4 1.09 x 10^4	4.70 x 10^4 4.26 x 10^4 4.68 x 10^4 4.55 x 10^4	1.20 x 10^5 1.14 x 10^5 1.21 x 10^5 1.18 x 10^5	9.64 x 10^5 7.89 x 10^5 9.67 x 10^5 9.07 x 10^5	9.6 9.8 9.8
Solvent Control       R1                           R2                           R3                              Mean	7.4 7.4 7.4	1.09 x 10^4 1.05 x 10^4 1.04 x 10^4 1.06 x 10^4	4.01 x 10^4 3.72 x 10^4 4.50 x 10^4 4.08 x 10^4	1.28 x 10^5 1.23 x 10^5 1.23 x 10^5 1.25 x 10^5	8.14 x 10^5 7.97 x 10^5 8.53 x 10^5 8.21 x 10^5	9.8 10.0 9.9
0.80                     R1                           R2                           R3                          R4                         R5                       R6                         Mean	7.3 7.3 7.3 7.3 7.3 7.3	9.66 x 10^3 9.78 x 10^3 9.04 x 10^3 9.88 x 10^3 9.23 x 10^3 9.29 x 10^3 9.48 x 10^3	3.58 x 10^4 3.56 x 10^4 4.61 x 10^4 3.66 x 10^4 3.18 x 10^4 3.67 x 10^4 3.71 x 10^4	1.31 x 10^5 1.16 x 10^5 1.22 x 10^5 1.14 x 10^5 1.19 x 10^5 1.22 x 10^5 1.21 x 10^5	8.85 x 10^5 8.62 x 10^5 7.56 x 10^5 8.26 x 10^5 7.81 x 10^5 8.72 x 10^5 8.30 x 10^5	9.7 9.5 9.5 9.6 9.5 9.7

 

Table 3                Inhibition of Growth Rate and Biomass

Nominal Concentration (mg/l)	Area Under Curve at 72 h	% Inhibition	Growth Rate (0 - 72 h)	% Inhibition
Control	1.42 x 10^7	-	0.061	-
Solvent Control	1.32 x 10^7	-	0.060	-
0.80	1.32 x 10^7	0	0.062	[3]

Validity criteria fulfilled:

yes

Remarks:

according to the OECD 201 (adopted 1984; 2006).

Conclusions:

The following effect concentrations on algal growth based on the time-weighted mean measured test concentrations were obtained: 72h-EbC50 (biomass) > 0.011 mg/l, 72h-ErC50 (growth rate) > 0.011 mg/l, 72h-NOEC (biomass, growth rate) >= 0.011 mg/l.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Scenedesmus subspicatus.The method followed the OECD guideline 201 (1984) and in compliance with GLP. Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous solution of the test material at a limit concentration of 0.80 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 + 1°C. Samples were removed daily and cell concentrations determined for each control and treatment group.

The test concentration of 0.80 mg/l was the highest attainable test concentration that could be prepared due to the limited water solubility of the test material and with the maximum amount of auxiliary solvent permitted by the guideline. Preliminary solubility work indicated that a test concentration of 0.80 mg/l could be best obtained using a solution in dimethylformamide. It was therefore considered inappropriate to test at concentrations in excess of 0.80 mg/l given that a significant proportion of the test material would not have been in solution and therefore not bioavailable to the algae. In order to confirm that the correct dosing procedures were followed a sample was prepared in culture medium alone (no algal cells) at a concentration of 0.80 mg/l. Chemical analyses of this sample showed a measured test concentration of 116% of nominal indicating that the correct dosing procedures were followed. Chemical analysis of the test solutions at 0 hours showed measured concentrations ranging from 50% to 56% of nominal for uncentrifuged samples, and a value of 3% of nominal for centrifuged samples. This was in line with the recovery analyses, which indicated that the test material adsorbed to algal cells. A marked decline was shown in the measured test concentrations after 72 hours with values ranging from 23 -24% of nominal for the uncentrifuged samples and to less than 1% of nominal for the centrifuged samples. This decline was considered to be due to further adsorption of the test material to algal cells. It was considered justifiable to base the results on the time-weighted mean measured test concentrations (TWA) of the centrifuged samples in order to have the best estimate of actual dissolved test item in the test solution.

Results of the study showed that there were no differences between the treatment and control groups with respect to both, growth rate and biomass production. In conclusion, the derived effect concentrations for Scenedesmus subspicatus based on measured concentrations (TWA) gave a 72h-ErC50 and 72h-EbC50 value of > 0.011 mg/l and 72h-NOEC of >= 0.011 mg/l test item.

The study fulfilled the validity criteria of the OECD guideline 201 (adopted 1984; 2006) and the results of the study were considered reliable.

Description of key information

72h-EbC50 (Scenedesmus subspicatus, biomass) > 0.011 mg/L (TWA); 72h-ErC50 (Scenedesmus subspicatus, growth rate) > 0.011 mg/L; 72h-NOEC (Scenedesmus subspicatus, biomass/growth rate) >= 0.011 mg/L (TWA)

Additional information

Two studies were performed to assess the effect of the test on the growth of green algae. One of the studies was considered as a reliable key study and is explained below. The second study (supporting study) was performed according to the EU testing method 88/203/EEC and not considered reliable, because the study inter alia did not include analytical determination of the test item concentrations, which was considered crucial with regard to the low water solubility of the test substance (<0.05 mg/L, see IUCLID section 4). Derived nominal effect concentrations here were: 72h-EbC50 (biomass) = 1.2 mg/L; 72h-ErC50 (growth rate) = 1.9 mg/L and 72h-NOEC (cell number) = 0.4 mg/L.

KEY STUDY

A reliable study was performed according to OECD 201 (1984) and in compliance with GLP (RL1).

Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous solution of the test material at a limit concentration of nominal 0.80 mg/L for 72 hours under static test conditions. The limit concentration of 0.80 mg/l test item was the highest attainable test concentration that could be prepared due to the limited water solubility of the test material (<0.05 mg/L, see IUCLID section 4) and with the maximum amount of auxiliary solvent permitted by the guideline. As a significant decline was shown in the measured test concentrations after 72 hours, effect concentrations were based on the time-weighted mean measured test concentrations (TWA).

Results of the study showed that there were no differences between the treatment and control groups with respect to both, growth rate and biomass production. The derived effect concentrations for Scenedesmus subspicatus based on measured concentrations (TWA) gave a 72h-ErC50 and 72h-EbC50 value of > 0.011 mg/l and 72h-NOEC of >= 0.011 mg/l test item.

The study fulfilled the validity criteria of the OECD guideline 201 (adopted 1984; 2006) and the results of the study were considered reliable.