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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:
06 September to ****
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:
yes
Remarks:
see below
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The following deviations from study plan occurred:

- The study plan states that the test will be conducted at a temperature range of 18-22 °C, however, during the definitive test, temperatures of 23 °C were recorded. Given that no adverse effects were observed, this was considered not to have had an impact on the outcome or integrity of the test.

- The study plan states that 20 daphnids will be added to each test concentration (4 replicates of 5), however, during the definitive test, no daphnia were observed to be present in replicates 1 to 3 of the 50% v/v saturated solution test group at 24 or 48 hours. Although this was a deviation to the study plan, given that no toxicity was observed in the higher concentration of 100% v/v saturated solution, this was considered not to have had an impact on the outcome or integrity of the test.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
CAS RN: 15337-18-5
Physical state/Appearance: Yellow viscous liquid
Purity: 96.6%

Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Preparation of Test Samples
The test samples were thawed with the aid of sonication before being filtered through 0.45 µm cellulose acetate filters.


Vehicle:
no
Details on test solutions:
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters 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 test concentrations of 6.25, 12.5, 25 and 50 % v/v saturated solution.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia Magna Straus taken from in-house laboratory cultures, maintained in Elendt M7 medium in a temperature controlled room at 18 to 22 °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. 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:
other: Reconstituted water (ISO medium)
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.
Hardness:
250 mg/L as CaCO3
Test temperature:
22 ºC to 23 ºC
pH:
7.9 to 8.1
Dissolved oxygen:
8.4 to 8.7 mg O2/L
Nominal and measured concentrations:
Definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
Details on test conditions:
As in the range-finding test, 150 mL glass jars containing approximately 100 mL of test preparation were used. At the start of the test five 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 covered and maintained in a temperature controlled room maintaining the water temperature at 22 to 23 °C, with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

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

The test solutions were not renewed during the exposure period.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
other: (geom. mean) as zinc (equivalent to 0.071 mg/L as test item)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
other: (geom. mean) as zinc (equivalent to 0.071 mg/L as test item)
Basis for effect:
mobility
Details on results:
Measured test concentrations in the maximum tested saturation level at 0 and 48 hour during the definitive test were determined to be 0.0091 mg/L as zinc (equivalent to 0.085 mg/L as test item) and 0.0064 mg/L as zinc (equivalent to 0.059 mg/L as test item), respectively. The geometric mean measured test concentration was determined to be 0.0076 mg/L as zinc (equivalent to 0.071 mg/L as test item).

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

No immobilization was observed in 20 daphnids exposed to test concentrations up to and including test concentrations of 100% v/v saturated solution for a period of 48 hours. No sub-lethal effects of exposure were observed throughout the test.

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 at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
Results with reference substance (positive control):
Exposure of Daphnia magna to the reference item, potassium dichromate, gave the following results:

24-hour EC50:  0.61 mg/L;
48-hour EC50:  0.54 mg/L;
No Observed Effect Concentration (NOEC): 0.32 mg/L
Lowest Observed Effect Concentration (LOEC): 0.56 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

Nominal
Concentration
(% v/v Saturated Solution)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

6.25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

12.5

0

0

0

0

0

0

5 N

5 N

5 N

5 N

25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

50

0

*

*

*

0

0

5 N

*

*

*

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

 

Nominal
Concentration
(% v/v Saturated Solution)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

6.25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

12.5

0

0

0

0

0

0

5 N

5 N

5 N

5 N

25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

50

0

*

*

*

0

0

5 N

*

*

*

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

N = Normal

R1– R4= Replicates 1 to 4

*No daphnids observed, no impact as no effects observed at the higher concentration of 100% v/v saturated solution

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item, zinc bis (dipentyldithiocarbamate) (oil free), to the freshwater invertebrate Daphnia magna was investigated over a 48-hour period. The 48-hour EC50 was determined to be greater than 100% v/v saturated solution (>0.0076 mg/L as zinc; equivalent to >0.071 mg/L as test item). The No Observed Effect Concentration was 100% v/v saturated solution (0.0076 mg/L as zinc; equivalent to 0.071 mg/L as test item).
No toxic effects were observed at exposure concentrations up to and including the limit of saturation in the test medium.
Executive summary:

A study was performed to assess the acute toxicity of the test item to 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. A preliminary media preparation trial indicated that a saturated solution method of preparation was the most appropriate for this test item.

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution for 48 hours at a temperature of 22 ºC to 23 ºC under static test conditions.  Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. An initial test was conducted, however, an inconsistent immobilization pattern was observed hence the test was repeated.

 

Measured test concentrations in the maximum tested saturation level at 0 and 48 hour during the definitive test were determined to be 0.0091 mg/L as zinc (equivalent to 0.085 mg/L as test item) and 0.0064 mg/L as zinc (equivalent to 0.059 mg/L as test item), respectively. The geometric mean measured test concentration was determined to be 0.0076 mg/L as zinc (equivalent to 0.071 mg/L as test item).

 

Exposure of Daphnia magna to the test item gave EC50 values of greater than 100% v/v saturated solution (>0.0076 mg/L as zinc; equivalent to >0.071 mg/L as test item). This study showed that there were no toxic effects at exposure concentrations up to and including the limit of saturation in the test medium. The No Observed Effect Concentration was 100% v/v saturated solution (0.0076 mg/L as zinc; equivalent to 0.071 mg/L as test item).

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the geometric mean measured test concentrations gave a 48-Hour EC50 value of greater than 0.0076 mg/L as zinc (equivalent to 0.071 mg/L as test item).  The No Observed Effect Concentration was 0.0076 mg/L as zinc (equivalent to 0.071 mg/L as test item) mg/L.  This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.007 mg/L

Additional information