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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 December 2009 to 29 January 2010

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/I) of test material with reconstituted water at approximately 1500 rpm for 24 hours and then removing the undissolved test material by filtration through a 0.2 µm Sartorius Sartopore filter (initial approximate 500 ml discarded to pre-condition the filter) to give a saturated solution with a nominal test concentration of 85 mg/I.
A media preparation trial conducted in reconstituted water showed a measured test concentration of approximately 85 mg/I for a saturated solution of the test material. This value was expressed as the nominal concentration for all work conducted prior to the definitive test.

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:

Water samples were taken from the control and the 0.85, 2.7, 8.5, 27 and 85 mg/I test groups (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis.
Duplicate samples and samples of the 1.5, 4.7, 15 and 47 mg/I test groups were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Pre-study solubility work showed that whilst the test material was readily soluble in deionised reverse osmosis water ie purified water and ASTM media used for the Algal Inhibition test (Harlan Laboratories Ltd Project No: 0656/0417), in the test media required for the Acute Toxicity to Daphnia magna test (reconstituted water), a 100 mg/I solution could not be attained.

The use of solvent stock solutions of the test material in order to spike the test medium showed that a test concentration of 5.0 mg/I could be attained, however, initial solubility work indicated that a higher measured concentration could be attained using a saturated solution method of preparation.

Therefore a media preparation trial was conducted in order to determine the amount of dissolved test material in a saturated solution prepared under test conditions.

An amount of test material (2250 mg) was dispersed in 22.5 litres of reconstituted water and stirred using a propeller stirrer at approximately 1500 rpm at a temperature of 21°C to 23°C. This was conducted in duplicate and stirred for 24 and 48 hours. After the stirring periods, samples were taken and analysed after filtration through a 0.2 µm Sartorius Sartopore filter pre-conditioned with approximately 500 ml and 1000 ml of the test solution. Samples were also analysed after centrifugation (10000 or 40000 g for 30 minutes).

Range-finding test:
An amount of test material (1100 mg) was added to 11 litres of reconstituted water and stirred using a propeller stirrer at approximately 1500 rpm for 24 hours at a temperature of approximately 21°C. After stirring, any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (initial approximate 500 ml discarded) to give a saturated solution, of 85 mg/I (the highest concentration). Serial dilutions in reconstituted water from this saturated solution were made using reconstituted water to give the remaining test concentrations of 8.5, 0.85 and 0.085 mg/I.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Initial test:
Based on the results of the range-finding test the following test concentrations were assigned to the initial test: 8.5, 15, 27, 47 and 85 mg/I.
An amount of test material (1100 mg) was added to 11 litres of reconstituted water and stirred using a propeller stirrer at approximately 1500 rpm for 24 hours at a temperature of approximately 21°C. After stirring any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (initial approximate 500 ml discarded) to give a saturated solution of 85 mg/I (highest test concentration). Aliquots (50, 88, 159 and 276 ml) of the saturated solution were each added separately to a final volume of 500 ml of reconstituted water to give the remaining test concentrations of 8.5, 15, 27 and 47 mg/I respectively.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Immobilisation at all concentrations was observed during the initial test, therefore, the test was repeated using a revised concentration range in order to ensure that a No Observed Effect Concentration could be obtained.

Definitive test:
An amount of test material (1100 mg) was added to 11 litres of reconstituted water and stirred using a propeller stirrer at approximately 1500 rpm for 24 hours at a temperature of approximately 21°C. After stirring any undissolved test material was removed by filtration through a 0.2 µm Sartorius Sartopore filter (initial approximate 500 ml discarded) to give a saturated solution of 85 mg/I (the highest test concentration). Aliquots (10, 18, 32, 55, 100, 176, 318 and 553 ml) of the saturated solution were each added separately to a final volume of 1 litre of reconstituted water to give the remaining test concentrations of 0.85, 1.5, 2.7, 4.7, 8.5, 15, 27 and 47 mg/I respectively.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: 1st instar Daphnia magna
- Source: derived from in-house laboratory cultures. Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at 19°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.
- Age at study initiation (mean and range, SD): Adult
- Weight at study initiation (mean and range, SD): N/A
- Length at study initiation (length definition, mean, range and SD): N/A
- Valve height at study initiation, for shell deposition study (mean and range, SD): N/A
- Peripheral shell growth removed prior to test initiation: N/A
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before the initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were then removed from the cultures and used for testing.
- Feeding during test
- Food type: The daphnids received no food during the exposure period.
- Amount: NDA
- Frequency: NDA

ACCLIMATION
- Acclimation period: NDA.
- Acclimation conditions (same as test or not): Same as test conditions
- Type and amount of food: Mixed algae (predominantly Chlorella spp.)
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): NDA

QUARANTINE (wild caught)
- Duration: N/A
- Health/mortality: N/A

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

N/A

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/I as CaCO3.

Test temperature:

19 - 21 °C

pH:

7.8 - 7.9

Dissolved oxygen:

8.7 - 10.2 mg O2/l

Salinity:

NDA

Nominal and measured concentrations:

Measured concentrations: following a preliminary range-finding and an initial test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 0.85, 1.5, 2.7, 4.7, 8.5, 15, 27, 47 and 85 mg/I for 48 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml glass jars contaning approximately 200 ml of test preparation
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: NDA

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Stock Solutions
a) CaCI2.2H20 11.76 g/I
b) MgSO4.7H20 4.93 g/I
c) NaHCO3 2.59 g/I
d) KCI 0.23 g/l
ii) Preparation

An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/I as CaCO3.

- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature was recorded daily and dissolved oxygen concentrations and pH were recorded at the start and termination of the study.

OTHER TEST CONDITIONS
- Adjustment of pH: if necessary with NaOH or HCI
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: NDA

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS

Range finding study
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.085, 0.85, 8.5 and 85 mg/I.
No significant immobilisation was observed at the test concentrations of 0.085, 0.85 and 8.5 mg/I. However, significant immobilisation was observed at 85 mg/I.
Initial study
- Test concentrations: Based on the results of the range-finding test the following test concentrations were assigned to the initial test: 8.5, 15, 27, 47 and 85 mg/I.
- Results used to determine the conditions for the definitive study: Immobilisation at all concentrations was observed during the initial study, therefore, the test was repeated using a revised concentration range in order to ensure that a No Observed Effect Concentration could be obtained. Based on the results of the initial test the following test concentrations were assigned to the definitive test: 0.85, 1.5, 2.7, 4.7, 8.5, 15, 27, 47 and 85 mg/I.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL: 18 - 21 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL: 17 - 20 mg/L

Effect concentrations 3

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

8.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: zero immobilisation

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

8.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: zero immobilisation

Details on results:

- Behavioural abnormalities: None recorded
- Observations on body length and weight: NDA
- Other biological observations: None recorded
- Mortality of control: 0%
- Other adverse effects control: None recorded
- Abnormal responses: None recorded

Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 24 and 48 hours.
The results of the definitive test differ slightly to the results of the initial test which showed 20% immobilisation at 8.5 mg/I after 48 hours exposure. Given that the results are in line with the range-finding test, chemical analysis of the test concentrations was within the acceptable limits of 80% to 120% and a NOEC was obtained it was considered that the difference observed had no effect on the outcome or validity of the test.

Observations on test material solubility: The test media preparations were observed to be clear, colourless solutions throughout the duration of the test.

Physico-chemical measurements: Temperature was maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100 %. This was considered to be due to the presence of microscopic air bubbles in the media supersaturating the diluent and was considered not to have had an impact on the outcome or validity of the test as no adverse effects were observed in the control daphnids.

Verification of test concentrations: Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 83% to 86% of nominal value 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):

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No: 0039/1133) during the positive control are given below.

Analysis of the immobilisation data by the probit method (Finney, 1971) at 24 hours and the trimmed Spearman-Karber method (Hamilton et al, 1977) at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/I) 95% Confidence limits (mg/I)
24 0.84 0.72 - 0.97
48 0.65 0.58 - 0.72

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/I. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slope and its standard error of the response curve at 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/I (sd = 0.20).

Reported statistics and error estimates:

Evaluation of data (definitive test):
The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by the trimmed Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999).

Evaluation of data (positive control):
The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney, 1971) using the ToxCalc computer software package (ToxCalc, 1999) and at 48 hours by the trimmed Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Any other information on results incl. tables

Table 1: Cumulative Immobilisation Data in the Range-Finding Test

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.085	0	0
0.85	0	1*
8.5	0	1*
85	10	10

 * Immobilised daphnids considered not to be significant given that the observed immobilisation was only 10%.

 

Table 2: Cumulative Immobilisation Data in the Initial Test

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
8.5	0	0	0	0	2	2	4	20
15	4	4	8	40	10	8	18	90
27	10	8	18	90	10	10	20	100
47	10	10	20	100	10	10	20	100
85	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

 

Table 3: Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.85	0	0	0	0	0	0	0	0
1.5	0	0	0	0	0	0	0	0
2.7	0	0	0	0	0	0	0	0
4.7	0	0	0	0	0	0	0	0
8.5	0	0	0	0	0	0	0	0
15	1	1	2	10	1	2	3	15
27	10	10	20	100	10	10	20	100
47	10	10	20	100	10	10	20	100
85	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

 

Table 4: Cumulative Immobilisation Data in the Positive Control 

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0
0.56	1	1	2	10	3	2	5	25
1.0	7	7	14	70	10	10	20	100
1.8	10	10	20	100	10	10	20	100
3.2	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 18 mg/I with 95% confidence limits of 17-20 mg/I. The No Observed Effect Concentration at 48 hours was 8.5 mg/I.

Executive summary:

The 48–hr-acute toxicity of the test material to Daphnia Magna was studied under static conditions.  Daphnids were exposed to the test material at nominal concentrations of 0.85, 1.5, 2.7, 4.7, 8.5, 15, 27, 47 and 85 mg a.i./L in aqueous solution for 48 hr.  Mortality/immobilization and sublethal effects were observed daily.  The 48–hour EC₅₀ was 18 mg a.i./L (nominal).  The 48–hr NOEC based on mortality/immobilization was 8.5 mg a.i./L (nominal).