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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
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 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group from the bulk test preparation at 0 hour and from the pooled replicates at 48 hours for quantitative analysis. Samples were analyzed immediately after sampling.

Duplicate samples were taken and stored frozen for further analysis, if necessary.
Vehicle:
no
Details on test solutions:
Information provided by the Sponsor indicated the functional solubility of the test item in water to be 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 solubility of the test item under test conditions.
Experimental Preparation

Test concentrations for the definitive test were selected based on available ecotoxicity data. A nominal amount of test item (2500 mg) was dispersed in 5 liters of test water with the aid of ultrasonication for 30 minutes. After sonication any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 32, 10, 3.2, 1.0 and 0.32% v/v saturated solution. Due to the light sensitive nature of the test item, all test item preparation was performed under laboratory safety lighting.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations was verified by chemical analysis at 0 and 48 hours.

Due to the light sensitive nature of the test item, all test item preparation was performed under laboratory safety lighting and all sample bottles were shielded from the light.

Results

Nominal Loading Rate (mg/L) Measured Concentration (mg/L)
10 9.47
100 98.5
500 259
1000 257
2000 253


Discussion
It is evident from these results that the limit of saturation of the test media was achieved at a nominal loading rate of 500 mg/L. Loading rates in excess of 500 mg/L did not result in an increase in the dissolved test item concentration obtained.

Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 500 mg/L, dispersed with the aid of ultrasonication of 30 minutes prior to the removal of any undissolved test item by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded) to give a measured test concentration of approximately 260 mg/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20°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. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3.
Test temperature:
21 +/- 1 degC.
Nominal and measured concentrations:
Nominal test concentrations :
0.32, 1.0, 3.2, 10, 32 and 100 % v/v saturated solutions

Mean measured test concentrations :

Verification of Test Concentrations
Analysis of the freshly prepared test preparations at 0 hour showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method, determined to be 0.021 mg/L to 187 mg/L. Analysis of the old or expired test preparations at 48 hours showed measured test concentrations ranged from less than the LOQ to 192 mg/L. It was considered appropriate to base the results on the mean measured concentrations.

Lower measured concentrations were observed in this study compared to those measured in the preliminary media preparation trial. This difference in the functional solubility of the test item could be attributed to the differences in test media 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:
Exposure Conditions
In the definitive test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test (within 30 minutes of the test media being added to the vessels), 5 daphnids were placed in each test and control vessel at random. Four replicate test and control vessels were prepared.

The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room for 48 hours at approximately 21°C in darkness. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test item. The test preparations were not renewed during the exposure period.

Any immobilization or adverse reactions (abnormal behavior or appearance) 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 immobilized if they were unable to swim for approximately 15 seconds after gentle agitation. Water temperature and pH were recorded daily in one replicate for each test and control group throughout the test. Dissolved oxygen concentrations were recorded at the start and termination of the test.
Reference substance (positive control):
yes
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL 1.4 - 6.1 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
6.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration throughout the test was =3 mg/L in the control and test vessels.

Temperature was maintained at approximately 21°C throughout the test. While there were no treatment related differences for oxygen concentration, concentration dependent differences in pH were observed throughout the test.
Results with reference substance (positive control):
A positive control test was conducted between 05 March 2014 and 07 March 2014, with used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. Exposure conditions for the positive control were similar to those in the definitive test. The 48 hour EC50 was 0.71 mg/L. The NOEC, based upon less than 10% immobilization at this concentration, was 0.56 mg/L and the LOEC was 1.0 mg/L. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Cumulative Immobilization Data in the Definitive Test

Nominal Concentration
(% v/v Saturated Solution)

Mean Measured Test Concentration
(mg/L)

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

0.32

<LOQ

0

0

0

0

0

0

0

0

0

0

0

0

1.0

1.44

0

0

0

0

0

0

0

0

0

0

0

0

3.2

6.13

5

4

5

5

19

95

5

5

5

5

20

100

10

19

5

5

5

5

20

100

5

5

5

5

20

100

32

60

5

5

5

5

20

100

5

5

5

5

20

100

100

190

5

5

5

5

20

100

5

5

5

5

20

100


Validity criteria fulfilled:
yes
Conclusions:
The 48 hour EC50 of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione to Daphnia magna is 3.0 mg/L, based on mean measured concentrations. The NOEC is 1.4 mg/L, and the LOEC is 6.1 mg/L.
Executive summary:

In an OECD 202 study, conducted according to GLP, the 48 hour toxicity (EC50) of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione to aquatic invertebrates (Daphnia magna)is 3.0 mg/L, based on the mean measured test concentration.

Description of key information

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
3 mg/L

Additional information

Key Study:

In an OECD 202 study, conducted according to GLP, the 48 hour toxicity (EC50) of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione to aquatic invertebrates (Daphnia magna)is 3.0 mg/L, based on the mean measured test concentration (Envigo Research Limited, 2016d).