4-oxo-4-(p-tolyl)butyric acid adduct with 4-ethylmorpholine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10.04.1995 - 13.04.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

An amount of test material (200 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 200 mg/l stock solution. This stock solution was mixed with 1 litre of algal suspension to give the test concentration of 100 mg/l.
The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 hours.

Vehicle:

no

Details on test solutions:

For the purpose of the definitive study the test material was prepared by a direct dispersion in culture medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Scenedesmus subspicatus (Chodat) strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria. Cultures were maintained in the laboratory by the transfer of algal cells to fresh culture medium approximately once per week. The culture was maintained in the laboratory at a temperature of 21 +/- 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 +/- 1 °C

pH:

8

Salinity:

NH4Cl: 15 mg/l
MgCl2 x 6H2O: 12 mg/l
CaCl2 x 2H2O: 18 mg/l
MgSO4 x 7H2O: 15 mg/l
KH2PO4: 1.6 mg/l
FeCl3 x 6H2O: 0.08 mg/l
Na2EDTA x 2H2O: 0.1 mg/l
H3BO3: 0.185 mg/l
MnCl2 x 4H2O: 0.415 mg/l
ZnCl2: 3E-3
CoCl2 x 6H2O: 1.5E-3
CuCl2 x 2H2O: 1E-5
Na2NoO4 x 2H2O: 7E-3
NaHCO3: 50 mg/l

Nominal and measured concentrations:

With respect to benzenebutanoic acid, 4-methyl-γ-oxo:
Vessel 1-3:
0 hours: nominal: 100 mg/l; measured: 99.9 mg/l
72 hours: nominal: 100 mg/l; measured: 98.4 mg/l

Vessel 4-6:
0 hours: nominal: 100 mg/l; measured: 100 mg/l
72 hours: nominal: 100 mg/l; measured: 99.9 mg/l

With respect to 4-ethylmorpholine:
Vessel 1-3:
0 hours: nominal: 100 mg/l; measured: 83.2 mg/l
72 hours: nominal: 100 mg/l; measured: 94.4 mg/l

Vessel 4-6:
0 hours: nominal: 100 mg/l; measured: 86.3 mg/l
72 hours: nominal: 100 mg/l; measured: 94.4 mg/l

Details on test conditions:

Based on the result of the range-finding study a "limit test" was conducted for the definitive study at a test concentration of 100 mg/l to confirm that at the maximum test concentration given in the OECD/EEC test guidelines no effect on algal growth was observed.
250 ml glass conical flasks were used.
Six flasks each containing 100 ml of solution were prepared for the treatment group and three flasks for the control group.
At initioation of the study the culture contained a nominal cell density of 1E4 cells per ml. The culture conditions gave an algal suspension in log phase growth characterised by an absorbance of 0.822 (@665 nm). This suspension was diluted to an absorbance of 0.029 prior to use.
The flasks were covered with aluminium foil and incubated (Gallenkamp INR-401-010W) at 24 +/- 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm using a Jenway 6100 Spectrophotometer. The cell densities of the control cultures at 0, 24, 48 and 72 hours, were determined by direct counting with the aid of a haemocytometer to confirm that the absorbance values were sufficiently well correlated with cell density values to be used to monitor the growth of test cultures.
The pH of each control and test flask was determined at initiation of the study and after 72 hours exposure. The temperature within the incubator was recorded daily.
Water samples were taken from the control and the 100 mg/l test group (replicates R1-R3 and R4-R6 pooled) at 0 and 72 hours for quantitative analysis.
Evaluation of data:
The area under the curve is taken to be an index of growth and was calculated.
Percentage inhibition of growth at each test concentration was calculated by comparing the area under the test curve with that under the control curve.
The EC50 value with respect to biomass was determined by inspection of the area under the growth curve data after 72 hours.
The average maximum growth rate for each culture was also calculated, from the straight section of the growth curve.
Percentage reductions in growth rate were calculated. The EC50 value with respect to growth rate was determined by inspection of the growth rates for the period 0-24 hours.
A Students t-test was carried out on the area under the growth curve data at 72 hours for the control and 100 mg/l test concentration to determine any statistically significant differences between the test and control groups.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

It can be seen that the absorbance values are well correlated with cell density values and therefore it was considered justifiable to monitor the growth of the test cultures by absorbance values alone.
It is clear that neither the growth or the biomass of Scenedesmus subspicatus (CCAP 276/20) was affected by the presence of 100 mg/l of the test material over the 72 hour exposure period.
Accordingly the following results were determined from the data:
E(b)C50 (72 h): >100 mg/l
E(r)C50 (0-24 h): >100mg/l
where E(b)C50 is the test concentration that reduced biomass by 50% and E(r)C50 is the tesh concentration that reduced specific growth rate by 50%.
Statistical analysis of the area under the growth curve data was carried out for the control and 100 mg/l test group using a Students t-test. There were no statistically significant differences (P >= 0.05) between the control and 100 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) is given as >= 100 mg/l.
It was considered unnecessary and unrealistic to test atv concentrations in excess of 100 mg/l.
The data show that the cell concentration of the control cultures increased by a factor of 42 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 100 mg/l.
The pH values of the control and test cultures were observed to increase from pH 7.1 - 8.0 at 0 hours to pH 10.0 - 10.3 at 72 hours. This effect is considered to be due to the large number of cells in the log phase of growth respiring pxygen and producing carbonates and biscarbonates as part of photosynthesis/respiration which in solution give rise to alkaline conditions.
Temperature was maintained at 24 °C throughout the study.
Analysis of the test solutions at 0 and 72 hours showed the measured test concentrations to be in excess of the required 80% of nominal test concentration and so it was considered justifiable to estimate EC50 values in terms of the nominal test concentration only.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave EC50 values of greater than 100 g/l. Correspondingly the No Observed Effect Concentration was greater than or equal to 100 mg/l.

Executive summary:

A study was performed to assess the effect of the test material on the growth of Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No. 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a concentration of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 °C.

Samples of the algal populations were removed daily and absorbance values determined for each control and treatment group.

Exposure of Scenedesmus subspicatus to the test material gave EC50 values of greater than 100 mg/l and correspondingly the No Observed Effect Concentration was greater than or equal to 100 mg/l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

Analysis of the test solutions at 0 and 72 hours showed the measured test concentrations to be in excess of the required 80% of nominal concentrations and so the results are based on nominal test concentrations alone.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15.02.1995 - 18.02.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

An amount of test material (200 mg) was dispersed in culture medium and the volume adjusted to 1 litre to give a 200 mg/l stock solution. Dilutions were made from this stock solution to give further stock solutions of 20, 2 and 0.2 mg/l. An aliquot (100 ml) of each stock solution was mixed with 100 ml algal suspension to give the test concentrations of 100, 10, 1 and 0.1 mg/l.

Vehicle:

no

Details on test solutions:

The test material was dispersed directly in culture medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Scenedesmus subspicatus (Chodat) strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria. Cultures were maintained in the laboratory by the transfer of algal cells to fresh culture medium approximately once per week. The culture was maintained in the laboratory at a temperature of 21 +/- 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 +/- 1 °C

pH:

8

Salinity:

NH4Cl: 15 mg/l
MgCl2 x 6H2O: 12 mg/l
CaCl2 x 2H2O: 18 mg/l
MgSO4 x 7H2O: 15 mg/l
KH2PO4: 1.6 mg/l
FeCl3 x 6H2O: 0.08 mg/l
Na2EDTA x 2H2O: 0.1 mg/l
H3BO3: 0.185 mg/l
MnCl2 x 4H2O: 0.415 mg/l
ZnCl2: 3E-3
CoCl2 x 6H2O: 1.5E-3
CuCl2 x 2H2O: 1E-5
Na2NoO4 x 2H2O: 7E-3
NaHCO3: 50 mg/l

Nominal and measured concentrations:

0.1 mg/l
1 mg/l
10 mg/l
100 mg/l

Details on test conditions:

The range-finding study was conducted by exposing Scenedesmus subspicatus cells to a series of nominal test concentrations of 0.1, 1, 10 and 100 mg/l for a period of 72 hours.
The study was conducted in 250 ml glass conical flasks loosely covered with aluminium foil to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. The test material was dispersed directly in culture medium.
At the start of the range-finding study a sample of each test and control culture was removed and the absorbance at 665 nm determined. The flasks were then covered with aluminium foil and incubated (Gallenkamp INR - 401-010W) at 24 +/- ^°C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 hours.
After 72 hours the absorbance of each flask was determined.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Validity criteria fulfilled:

yes

Conclusions:

The absorbance values showed no effect on growth at all concentrations tested.

Executive summary:

Based on this information a single test concentration of six replicates, of 100 mg/l was selected for the definitive study. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC test guidelines no effect on growth was observed.

Description of key information

The effect of the test material on the growth of Scenedesmus subspicatus has been investigated and gave EC50 values of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was greater than or equal to 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