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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
Deviations stated in 'Principles of method if other than guideline'
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
Deviations stated in 'Principles of method if other than guideline'
Principles of method if other than guideline:
Deviation No.1
The study plan states that all samples were to be analyzed immediately after sampling. However, due to instrumentation problems, even though all samples were provided for immediate analysis, the 0, 24, 48 and 72 hour samples were stored frozen prior to immediate analysis with the 96 hour samples. Given that the results from the 96-Hour samples were consistent with those of all other time points, this deviation was not considered to have had an adverse effect on the outcome of the test.

Deviation No.2
The study plan states that the temperature range in the study is recommended to be 21-25°C. The fish in this study, however, were maintained at 20 ±1°C and, on occasion, for an unknown reason, the temperatures recorded in the study decreased to 19°C. Given that the test temperature remained constant within the ± 2°C in accordance with the guidelines and no adverse effects were observed in the control or test group, this temperature deviation was considered not to have had an adverse effect on the outcome or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples (200 mL) were taken from the control and the 100% v/v saturated solution test group from freshly prepared at 0, 24, 48 and 72 hours and from old solutions at 24, 48, 72 and 96 hours for quantitative analysis.

Duplicate samples were taken and stored frozen for further analysis, if necessary.
Whilst all samples were provided for immediate analysis, due to an instrumental malfunction, the 0, 24, 48 and 72-hour test samples were stored frozen prior to analysis with the 96-hour samples.


Duplicate samples were taken and stored frozen for further analysis, if necessary.
Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial

Information provided by the Sponsor indicated the functional solubility of the test item in water is less than 320 mg/L. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore, a media preparation trial was conducted in order to determine the functional solubility of the test item under test conditions.
Experimental Preparation
A nominal amount of test item (11 g) was dispersed in 22 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 1 hour. After 1 hour the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. The pH of the 100% v/v saturated solution was measured and adjusted to that of the control solution prior to exposure of the fish. Dilutions were then prepared from the 100% v/v test concentration to produce the remaining test concentrations. The test concentrations were mixed with a flat bladed stirrer for 1 minute to ensure adequate mixing and homogeneity. Due to the light sensitive nature of the test item, the test item was prepared under laboratory safety lighting.
Test organisms (species):
Pimephales promelas
Details on test organisms:
The test was carried out using juvenile fathead minnow (Pimephales promelas). Fish were maintained in house since 31 October 2014. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish used in the definitive test were acclimatized to test conditions from 01 December 2014 to 08 December 2014. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at approximately 20°C with a dissolved oxygen content of greater than or equal to 9.3 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial ZM400 food which was discontinued approximately 24 hours prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 2.4 cm (sd = 0.1) and a mean weight of 0.19 g (sd = 0.02) at the end of the definitive test. Based on the mean weight value this gave a static loading rate of
0.07 g bodyweight/liter.

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure.

The length and weight of the control fish were determined at the end of the test. Based on the mean weight value this gave a static loading rate of 0.07 g bodyweight/liter.
Test temperature:
21°C
pH:
pH 8.1
The pH of the 100% v/v saturated solution was adjusted to that of the control solution using 1.0 M NaOH prior to exposure of the fish.
Dissolved oxygen:
8.7 mg O2/L
Nominal and measured concentrations:
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration, no mortalities or sub-lethal effects of exposure were observed.

The mean measured test concentration was determined to be:

Nominal Test Concentration (% v/v Saturated Solution): 100%

Mean Measured Test Concentration (mg/L): 454 mg/L

Higher measured concentrations were observed in this study compared to those measured in the preliminary media preparation trial. This difference in functional solubility of the test item could be attributed to the differences in test media and/or pH adjustment required in this study, but was considered not to have had an adverse effect on the outcome or integrity of the study.
Details on test conditions:
Test Water:
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

Exposure Conditions:
25-30 liter glass exposure vessels, each containing 20 liters of media, were used for the range-finding test and definitive limit test. At the start of the definitive limit test, seven fish were placed in each test vessel at random. The test vessels were then covered to reduce evaporation and maintained at approximately 20±1°C in a temperature controlled room in darkness for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure. The test vessels were labeled with the study number and all relevant information.

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

A semi-static test regime was employed in the test, involving a daily renewal of the test preparations to prevent the build upbuild-up of nitrogenous waste products extremely toxic to fish (ammonia).
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 454 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 454 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
No mortalities were observed in the control fish or fish exposed to a mean measured concentration of 454 mg/L for a period of 96 hours.

No sub lethal effects of exposure were observed in the control fish or fish exposed to a test concentration of 454 mg/L for a period of 96 hours.
Sublethal observations / clinical signs:

Range-finding Test

Cumulative mortality data from the exposure of fathead minnow to the test item during the range-finding test were monitored. The results showed no mortalities at the test concentrations of 1.0, 10 and 100% v/v saturated solution. There were no sub-lethal effects of exposure during the range-finding test.

 

Chemical analysis of the test solutions at 0 hours showed measured concentrations of test item to range from 3.2 to 374 mg/L and at 24 hours to range from 3.4 to 401 mg/L indicating that the test item was stable under test conditions.

 

Based on this information, a single test concentration of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration, no mortalities or sub lethal effects of exposure were observed.

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was = 60% of ASV (= 5.5 mg O2/L) in the control and test vessels.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item, 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione, to the freshwater fish fathead minnow (Pimephales promelas) was investigated over 96 hours and, based on the mean measured test concentration, the 96-Hour LC50 was determined to be greater than 454 mg/L. This study shows that there were no toxic effects of 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione on fish up toat itsthe limit concentration tested, 454 mg/Lof solubility.

This study shows that there were no toxic effects at the limit concentration tested, 454 mg/L.
Executive summary:

In an OECD 203 study, according to GLP, the 96 hour toxicity (LC50) of 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione to fish (Pimephales promelas), based on the mean measured test concentration, is greater than 454 mg/L.

Description of key information

Key value for chemical safety assessment

Additional information

Key Study:

In an OECD 203 study, according to GLP, the 96 hour toxicity (LC50) of 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione to fish (Pimephales promelas), based on the mean measured test concentration, is greater than 454 mg/L (Envigo Research Limited, 2016c).