2,4-Thiophenedicarbonitrile, 5-amino-3-methyl-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 November 2009 to 4 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

EC Number:

610-868-8

Cas Number:

52603-48-2

Molecular formula:

C7N3H5S

IUPAC Name:

5-amino-3-methylthiophene-2,4-dicarbonitrile

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration and stability of the test material in the test solutions were verified by chemical analysis.
- Sampling method: Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken at 0 and 72 hours and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Range finding test:

An amount of test material (100 mg) was dissolved in culture medium with the aid of high shear mixing at approximately 7500 rpm for 45 minutes and the volume adjusted to 1 litre to give a 100 mg/I stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 10, 1.0 and 0.10 mg/I. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (3.5 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/I.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Definitive test:

An amount of test material (100 mg) was dissolved in culture medium with the aid of high shear mixing at approximately 7500 rpm for 45 minutes and the volume adjusted to 1 litre to give a 100 mg/I stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 50, 25, 12.5 and 6.25 mg/I. An aliquot (500 ml) of each of the stock solutions was separately inoculated with algal suspension (4.7 ml) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/I.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): NDA
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C.

ACCLIMATION
- Acclimation period: NDA
- Culturing media and conditions (same as test or not): The culture medium used for the studies was the same as that used to maintain the stock culture.
- Any deformed or abnormal cells observed: NDA

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

NDA

Test temperature:

24 ± 1 °C

pH:

Test vessels: 7.0 - 7.5
The pH values of the control cultures were observed to increase from pH 7.1 at 0 hours to pH 7.5 - 7.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

NDA

Salinity:

NDA

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 87% to 89% of nominal and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with polyurethane foam bungs
- Material, size, headspace, fill volume: 250 ml glass conical flasks each containing 100 ml of test preparation
- Aeration: NDA
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 4.15 x 10E3 cells per ml
- Control end cells density: 1.05 x 10E5 cells per ml
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

NaNO3 25.5 mg/I
MgCI2.6H20 12.164 mg/I
CaCI2.2H20 4.41 mg/I
MgSO4.7H20 14.7 mg/I
K2HP04 1.044 mg/I
NaHCO3 15.0 mg/I
H3BO3 0.1855 mg/I
MnCI2.4H20 0.415 mg/I
ZnCI2 0.00327 mg/I
FeCI3.6H20 0.159 mg/I
CoCI2.6H20 0.00143 mg/I
Na2MoO4.2H20 0.00726 mg/I
CuCI2.2H20 0.000012 mg/I
Na2EDTA.2H20 0.30 mg/I
Na2SeO3.5H20 0.000010 mg/I

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals: NDA
- Pesticides: NDA
- Chlorine: NDA
- Alkalinity: NDA
- Ca/mg ratio: NDA
- Conductivity: NDA
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of each control and test flask was determined at initiation of the study and after 72 h exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: NDA
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI
- Photoperiod: continuous illumination
- Light intensity and quality: light intensity approximately 7000 lux provided by warm white lighting (380 - 730 nm)
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: samples were taken at 0, 24, 47 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Chlorophyll measurement: none
- Other: The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a VVTW pH 320 pH meter. The temperature within the incubator was recorded daily.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: NDA
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 mg/I. However, growth was observed to be reduced at 100 mg/I.
Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/I were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate 95% confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

90 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: It was not possible to calculate 95% confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

The following data show that the cell concentration of the control cultures increased by a factor of 25 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.

Mean cell density of control at 0 hours : 4.15 x 103 cells per ml
Mean cell density of control at 72 hours : 1.05 x 105 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 9% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.The following data show that the cell concentration of the control cultures increased by a factor of 25 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.

Mean cell density of control at 0 hours : 4.15 x 103 cells per ml
Mean cell density of control at 72 hours : 1.05 x 105 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 9% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50 mg/I, however no intact cells were observed to be present in the test cultures at 100 mg/I.
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 6.25, 12.5, 25 and 50 mg/I test cultures were observed to be pale green dispersions whilst the 100 mg/I test cultures were observed to be clear colourless solutions.
Temperature was maintained at 24 ± 1°C throughout the test.
The pH values of the control cultures were observed to increase from pH 7.1 at 0 hours to pH 7.5 - 7.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 87% to 89% of nominal and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 0039/1088) used potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/I.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:
ErC50 (0 - 72 h): 0.79 mg/l*
EyC50 (0 - 72 h): 0.30 mg/I, 95% confidence limits 0.27 - 0.34 mg/I

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/I
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/I

Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/I
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/I

The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant decreases in growth rate between the control, 6.25, 12.5, 25 and 50 mg/I test concentrations (P>=0.05), however the 100 mg/I test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 50 mg/I. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100 mg/I.
Statistical analysis of the yield data was carried out as for the growth rate data. There were no statistically significant decreases in yield between the control, 6.25, 12.5, 25 and 50 mg/I test concentrations (P>=0.05), however the 100 mg/I test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 50 mg/I. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100 mg/I.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see above

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results:
EC50 (mg/l) 95% Confidence Limits (mg/I) NOEL (mg/I) LOEC (mg/I)
Growth Rate 100 * 50 100
Yield 90 * 50 100

* It was not possible to calculate 95% confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits.

Executive summary:

In a 72 hour acute toxicity study, the cultures of the green alga Desmodesmus subspicatus were exposed to the test material at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L under static conditions in accordance with Method C.3 of Commission Directive 92/69/EEC. The NOEC and EC50 values based on yield were 50 and 90 mg/L (nominal) respectively.