1,4-Dihydroxy-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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Definitive test: 100mg/L

- Sampling method:
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours. Water samples were taken from the control and the 100 mg/L test group (replicates R1 - R3 pooled and R4 - R6 pooled).

- Sample storage conditions before analysis:
Duplicate samples were taken at each occasion and stored at approximately -20 ºC for further analysis if necessary.

Vehicle:

no

Details on test solutions:

RANGE-FINDING TEST:
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Scenedesmus subspicatus cells to a series of nominal test concentrations of 1.0, 10 and 100 mg/L for a period of 72 hours.

The test was conducted in 250 mL glass conical flasks loosely covered with aluminium foil to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.

An amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 200 mg /L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 20 and 2.0mg/L. An aliquot (100 mL) of each of the stock solutions was separately inoculated with algal suspension (100 mL) to give the required test concentrations of 1.0, 10 and 100 mg/L..

In order to ensure homogeneity of the samples the volumetric flasks were inverted several times.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter Multisizer II Particle Counter. The flasks were then covered with aluminium foil and incubated (Gallenkamp INR - 401-010W) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) and constantly shaken at approximately 100 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter Multisizer II Particle Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 96 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20°C prior to analysis.

DEFINITIVE TEST:
Based on the results of the range-finding test a limit test was conducted for the definitive study at a test concentration of 100 mg/l.

Experimental Preparation:
The test material was prepared by a direct solution in culture medium
An amount of test material (100mg) was dissolved in culture medium and the volume adjusted to 500ml to give a 200mg/l stock solution. This stock solution was inoculated with algal suspension (500ml) to give the required concnetration of 100mg/l.

In order to ensure homogeneity of the sample the volumetric flask was inverted several times.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test Species
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, The Ferry House, Far Sawrey, Ambleside, Cumbria. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory under constant aeration and constant illumination (intensity approximately 7000 lux) at 21 ± 1 °C.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable.

Test temperature:

Temperature was maintained at 24 ± 1°C throughout the study.

pH:

The pH of each control cultures and test flask was determined at initiation of the test and after 72 hours exposure.

Nominal and measured concentrations:

Range-finding Test: Nominal concentrations of 1.0, 10 and 100 mg/L
Definitive test: Nominal concentration, Limit test 100mg/l.

Details on test conditions:

DEFINITIVE TEST EXPOSURE CONDITIONS:
250 mL glass conical flasks were used. Six flasks each containing 100mL of solution were prepared for the treatment group and three flasks each for the control.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.31 x 10E6 cells per mL. This suspension was diluted to a cell density of 2.47 x 10E4 cells per mL prior to use. At initiation of the study the culture conatained a nominal cell density of 10E4 cells per ml.

The flasks were plugged with polyurethane foam bungs and incubated (Gallenkamp INR-401-0104) at 24 ± 1°C. under continuous illumination (intensity approximately7000 lux) and constantly shaken at approximately 100 rpm for 72 hours.


GROWTH MEDIUM
Culture medium: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The culture medium is defined below:
NaNO3: 25.5 mg/l
MgCl2.6H2O: 12.164 mg/l
CaCl2.2H2O: 4.41 mg/l
MgSO4.7H2O: 14.7 mg/l
K2HPO4 1.044: mg/l
NaHCO3 15.0: mg/l
H3BO3 0.1855: mg/l
MnCl2.4H2O: 0.415 mg/l
ZnCl2: 0.00327 mg/l
FeCl3.6H2O: 0.159 mg/l
CoCl2.6H2O: 0.00143 mg/l
Na2MoO4.2H2O: 0.00726 mg/l
CuCl2.2H2O: 0.000012 mg/l
Na2EDTA.2H2O: 0.30 mg/l
Na2SeO3.5H2O: 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. The prepared media was sterilised by 0.2μm membrane filtration and stored in darkness.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Samples were taken at 0, 24. 48 and 72 hours and the cell densities determined by using a Coulter Multisizer II Particle Counter.

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material and oxidation products

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material and oxidation products

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material and oxidation products

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material and oxidation products

Basis for effect:

biomass

Details on results:

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

Physico-chemical measurements
See attached background information.
The pH values of the control cultures were obserevd to increase from pH 7.6 at 0 hours to pH 9.5 at 72 hours. This increase is 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 required for photosynthesis and growth will 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 the recommended EEC guidelines (1.5 pH units after 72 hours). This 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 exceeeded the validation criteria.

Range-finding Test

The cell counts and percentage inhibition of growth values are given in the attached background information.

The results showed no effect on growth at the test concentrations of 1.0, 10 and 100 mg/l

Based on this information a single test concentration (six replicates), of 100 mg/l was selected for the definitive study.

Definitive study

Cell density values determined at each sampling time and pH values at 0 and 72 hours, inhibition of growth rate and biomass, growth curve are given in the attached background information.

Neither the growth rate or the biomass of Scenedesmus subspicatus were affected by the test material over the 72 hour exposure period.

Accordingly the following results were determined from the data:

EbC50 (72 h) : >100mg/l

ErC50 (0 -72 h) : >100mg/l

There were no statistically significant differences (p ≥ 0.05), between the control and the 100 mg/l test group and therefore the No Observed Effect Concentration (NOEC) is 100 mg/l

Validation Criteria:

The cell concentration of the control cultures increased by a factor of 53 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Verification of test concentrations

See attached background information

Data indicated that the test material decomposes in solution when exposed to air, therefore samples taken from the definitive study were analysed for both the parent test material and the oxidation product at 0 and 72 hours.

Analysis of the test solutions at 0 hours showed the measured concentration of the parent test material to be 93mg/l and 94 mg/l (R1 - R3 and R4 - R6 pooled respectively) and measured test concentrations of the oxidation product to be 3.1 mg/l and 3.3 mg/l

(R1 - R3 and R4 - R6 pooled respectively).

At 72 hours analysis of the test solutions showed the measured concentration of the parent test material to be 46mg/l and 43 mg/l (R1 - R3 and R4 - R6 pooled respectively) and the oxidation product to be 39 mg/l and 51 mg/l (R1 - R3 and R4 - R6 pooled respectively).

Given that the test material was shown to decompose over the study period, toxicity cannot be attributed to the parent test material alone but to the test material / oxidation product as a whole. As such EC50 values cannot be calculated based on the measured test concentration of the parent material or oxidation product and therefore the EC50 values given have been calculated based on nominal test concentration only.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Scenesdesmus subspicatus has been investigated over a 72-Hour period and gave the following results:

Growth Rate:
72 h EC50: >100 mg/L
72 h NOEC: 100 mg/L

Biomass:
72 h EC50: >100 mg/L
72 h NOEC: 100 mg/L

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Scenesdesmus subspicatus.

The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1984) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC.

Following a preliminary range-finding test, Scenesdesmus subspicatus was exposed to an aqueous solution of the test item at concentrations of 100mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

Exposure of Scenesdesmus subspicatus to the test item gave an EC50 value >100 mg/l and correspondingly the NOEC was 100 mg/l.

Analysis of the test solutions at 0 hours showed the measured concentration of the parent test material to be 93mg/l and 94 mg/l (R1 - R3 and R4 - R6 pooled respectively) and measured test concentrations of the oxidation product to be 3.1 mg/l and 3.3 mg/l

(R1 - R3 and R4 - R6 pooled respectively).

At 72 hours analysis of the test solutions showed the measured concentration of the parent test material to be 46mg/l and 43 mg/l (R1 - R3 and R4 - R6 pooled respectively) and the oxidation product to be 39 mg/l and 51 mg/l (R1 - R3 and R4 - R6 pooled respectively).

Given that the test material was shown to decompose over the study period, toxicity cannot be attributed to the parent test material alone but to the test material / oxidation product as a whole. As such EC50 values cannot be calculated based on the measured test concentration of the parent material or oxidation product and therefore the EC50 values given have been calculated based on nominal test concentration only.

Description of key information

Exposure of Scenesdesmus subspicatus to the test item gave 72 hour EC50 values greater than 100 mg/L. The 72 hour No Observed Effect concentration was 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Scenesdesmus subspicatus.

The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1984) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC.

Following a preliminary range-finding test, Scenesdesmus subspicatus was exposed to an aqueous solution of the test item at concentrations of 100mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

Exposure of Scenesdesmus subspicatus to the test item gave an EC50 value >100 mg/l and correspondingly the NOEC was 100 mg/l.

Analysis of the test solutions at 0 hours showed the measured concentration of the parent test material to be 93mg/l and 94 mg/l (R1 - R3 and R4 - R6 pooled respectively) and measured test concentrations of the oxidation product to be 3.1 mg/l and 3.3 mg/l

(R1 - R3 and R4 - R6 pooled respectively).

At 72 hours analysis of the test solutions showed the measured concentration of the parent test material to be 46mg/l and 43 mg/l (R1 - R3 and R4 - R6 pooled respectively) and the oxidation product to be 39 mg/l and 51 mg/l (R1 - R3 and R4 - R6 pooled respectively).

Given that the test material was shown to decompose over the study period, toxicity cannot be attributed to the parent test material alone but to the test material / oxidation product as a whole. As such EC50 values cannot be calculated based on the measured test concentration of the parent material or oxidation product and therefore the EC50 values given have been calculated based on nominal test concentration only.