Molybdenum, bis(dibutylcarbamodithioato)di-μ-oxodioxodi-, sulfurized

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 July 2016 to 12 July 2018

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)

Specific details on test material used for the study:

CAS RN: 68412-26-0
Purity: This substance has an Unknown or Variable
composition, is a Complex reaction product, or a Biological material (UVCB)
Description: Yellow powder

Analytical monitoring:

yes

Details on sampling:

Samples from the control and the 100 mg/L loading rate WAF test group were taken from the freshly prepared bulk test preparation at 0 and 24 hours and from the expired pooled replicates (replicates R1 – R4) at 24 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.

Preparation of Calibration Standards
The test item (nominal 20 mg) was dissolved in acetonitrile (200 mL) to prepare a stock solution with a nominal concentration of 100 mg/L. This stock solution was further diluted with acetonitrile to obtain a nominal 1.0 mg/L calibration standard. A duplicate calibration standard was similarly prepared at 1.0 mg/L. These duplicate calibration standards were used to determine the recovery and test sample concentrations.

Preparation of Test Samples
The test samples were thawed in a water bath. An aliquot (0.5 mL) of formic acid was added to the test samples (100 mL). The test item was extracted from the test samples using a solid phase extraction cartridge (strata C8, 500 mg/ 3 mL). The cartridge was pre-conditioned with 10 mL of acetonitrile and 10 mL of water. The samples were drawn through the cartridge under reduced pressure. Subsequently, the cartridge was eluted with 20 mL of acetontrile into a 20 mL volumetric and made up to the mark with acetonitrile.

Vehicle:

no

Details on test solutions:

Details on test solutions
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

A nominal amount of test item (500 mg) was added to the surface of 5 liters of test water to give the 100 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. After the standing period, a wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF.

Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: In-house
- Age: Less than 24 hours old
- Food: Culture was fed with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
- Frequency: Daily

Test type:

semi-static

Water media type:

other: Reconstituted water (ISO medium)

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

21 to 22 ºC

pH:

7.4 to 7.9

Dissolved oxygen:

8.1 to 9.1mg O2/L

Nominal and measured concentrations:

Nominal:
100 mg/L

Measured:
Chemical analysis of the fresh test preparations at 0 and 24 hours showed that measured test concentrations of 0.047 mg/L and 0.073 mg/L were obtained respectively. Chemical analysis of the aged test preparations at 24 and 48 hours showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method which was determined to be 0.014 mg/L.

Details on test conditions:

TEST SYSTEM
Test solutions were prepared in the same manner as described for the range-finding test. Glass jars (150 mL) containing approximately 100 mL of test solution were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained concurrently under identical conditions but was not exposed to the test item. Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-hour old test media into the fresh test media.

OTHER TEST CONDITIONS
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations, while the measurements taken prior to the test media renewal and at test termination after 48 hours, represent those of the 24-hour old test preparations. The pH and dissolved oxygen concentration, temperature and light intensity were measured. The appearance of the test media was recorded daily.

RANGE-FINDING STUDY
Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L. Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. In the range-finding test, 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded. The control group was maintained concurrently under identical conditions but was not exposed to the test item. A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Immobilization Data
No immobilization was observed in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Sub-Lethal Effects
No sub-lethal effects of exposure were observed throughout the definitive test.

Results with reference substance (positive control):

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item, potassium dichromate, gave the following results:

24-hour EC50:  0.83 mg/L; 95% confidence limits 0.70 – 0.98 mg/L
48-hour EC50:  0.64 mg/L; 95% confidence limits could not be determined.
No Observed Effect Concentration (NOEC): 0.56 mg/L
Lowest Observed Effect Concentration (LOEC): 1.0 mg/L

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

Cumulative Immobilization Data and Observations in the Definitive Test

.	24 Hours
.	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)					Observations
Nominal Loading Rate (mg/L)	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

 

.	48 Hours
.	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)					Observations
Nominal Loading Rate (mg/L)	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

R1 – R4 = Replicates 1 to 4

N = No sub-lethal effects observed

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Executive summary:

A study was performed to assess the acute toxicity of the test item to the freshwater invertebrate, Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No. 202, "Daphnia sp., Acute Immobilisation Test" and Method C.2 of Commission Regulation (EC) No. 440/2008.

Due to the low aqueous solubility and complex nature of the test item, the test solutions were prepared as Water Accommodated Fractions (WAF).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 ºC to 22 ºC under semi-static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Analysis of the freshly prepared test solutions at 0 and 24 hours showed that measured test concentrations of 0.047 mg/L and 0.073 mg/L were obtained, respectively. Chemical analysis of the aged test solutions at 24 and 48 hours showed that measured concentrations were less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.014 mg/L.

Given that the toxicity in this study cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Daphnia magna to the test item resulted in EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L loading rate WAF.

Description of key information

Under the conditions of the study, exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L