Nickel bis(dibutyldithiocarbamate)

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Remarks:

Study planned. Difficulties with development of analytical method resulted in delays.

Adequacy of study:

key study

Study period:

The study will be available in November 2019

Reliability:

other: Study ongoing

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

October 2012

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E 1193-97: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests

Version / remarks:

2012

GLP compliance:

yes

Specific details on test material used for the study:

The information on specific details on the test material used for the study is not available yet.

Analytical monitoring:

yes

Details on sampling:

Water samples will be collected from each treatment and control group prior to the start of exposure to confirm the operation of the diluter system. More than one pretest sampling interval may be conducted if needed. Samples of stock solutions may be collected for analysis as needed during the test to confirm concentrations being delivered to the diluter system. Water samples will be collected from one replicate test chamber in each treatment and control group at the beginning of the test, approximately weekly during the test, and at the end of the test to determine concentrations of the test substance. Sampling typically will rotate among the replicates in each group at each sampling interval. In the event that 100% immobility occurs in any treatment, final sampling of that treatment will be conducted on that day. Samples will be collected at mid-depth, and typically will be processed immediately for analysis. If necessary, the samples will be placed in an appropriate storage container (e.g., glass or polypropylene bottle) and stored under the appropriate conditions (e.g., refrigeration or ambient) until analyzed. If a precipitate is observed in any test chambers, the samples will be centrifuged prior to analysis.

Vehicle:

not specified

Details on test solutions:

The test substance will be administered to the test organism in water. This route of administration was selected because it represents the most likely route of exposure to aquatic organisms. The test substance typically will be mixed in dilution water or reverse-osmosis water to form one or more stock solutions. Additional stock solutions may be prepared by dilution from a primary stock. Aliquots of the stock solutions will subsequently be mixed with dilution water in the diluter system to prepare the test solutions. The test substance will be delivered using solvent-free methods, if practical. If necessary to facilitate delivery of the test substance, stock solutions may be prepared in an organic solvent. Aliquots of the solvent stock solutions would subsequently be mixed with dilution water in the diluter system to prepare the test solutions. Acceptable solvents include, but are not limited to, dimethylformamide, triethylene glycol, methanol, ethanol, or acetone. Organic solvents will be reagent grade or better. If an organic solvent is required, a solvent control will be included in the experimental design along with a negative (dilution water) control group. The concentration of the organic solvent in the test solution will be minimized when possible (e.g., 20 µL/L), but will not exceed 100 µL/L. The organic solvent concentration in the solvent control and all exposure groups will be equal. The specific methodology used to prepare the test solutions will be documented in the raw data and summarized in the final report.

Test organisms (species):

Daphnia magna

Details on test organisms:

Parental-generation daphnids to be used in the test will be less than 24 hours old and will be obtained from cultures maintained at the test facility.

CULTURING
Daphnids received from a supplier to initiate cultures will be held for at least 21 days prior to the use of organisms for testing. Daphnids will be cultured in water from the same source and at approximately the same temperature as will be used during the test. Changes in water temperature will not exceed 1ºC in any 24-hour period during holding. Brood daphnids in the cultures producing neonates for the test will be held for at least 10 days prior to collection of the neonates for testing. A culture will not be used to provide neonates for the test if adults do not produce by Day 12 of holding, if adults do not produce an average of at least three young per adult per day over the 7 day period prior to the test, if > 10% of the culture stock die or show signs of disease or stress (e.g., exhibiting abnormal behavior) in the two-day period prior to test initiation, or if the culture contains ephippia. While the 10% rate for immobility and signs of disease or stress is higher than indicated in the OCSPP guideline, this rate is based upon past history of use in the laboratory, is a more reasonable rate for invertebrate species, and is more consistent with the allowable rate of control immobility during the test. Neonates will be obtained for testing from at least three individual adults that have already produced at least one prior brood. Neonates from daphnids that show signs of disease or stress will not be used as test organisms, and no daphnids used in the test will have been used in previous testing.

FEEDING
Daphnids in the cultures will be fed a mixture of yeast, cereal grass media, and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Raphidocelis subcapitata, at sufficient rates to support normal growth and development and to allow for asexual reproduction. Daphnids will be fed daily during the test (typically 2-3 times per day through Day 7 and 4 times per day until the last day of the test) using the same diet as used in the cultures. The multiple feedings each day are used to maintain sufficient feed in the flow-through system to support acceptable reproduction rates. The quantity and frequency of feedings will be documented in the raw data and summarized in the final report. Analyses will be performed at least once annually to determine the concentrations of selected organic and inorganic constituents of the YCT feed and results of the analyses will be summarized in the final report. Specifications for acceptable levels of contaminants in daphnid diets have not been established. However, there are no known levels of contaminants reasonably expected to be present in the diet that are considered to interfere with the purpose or conduct of the test.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (solvent control), 1.9, 3.8, 7.5, 15 and 30 µg/L (based on additional solubility work performed).

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers in the diluter system will be aquaria made of glass or of Teflon®-lined or non-lined stainless steel. Typically tests are conducted in 12-L glass aquaria filled with approximately 10 L of test solution, but the aquaria type, size and volumes may need to be changed to address the physical/chemical properties of the test substance. Each test chamber will contain one test compartment suspended in the water column. Test compartments typically will be constructed from glass beakers, approximately 6.5 cm in diameter and 12 cm in height. Nylon 220-µm mesh screens will cover two holes on opposite sides of each test compartment to permit test solution to flow in and out of the compartment while ensuring that the daphnids are submerged in at least 5 cm of test solution. The test compartment and test chamber type (e.g., glass), size and volume of test solution used in the test will be documented in the raw data and presented in the final report. Test chambers will be indiscriminately positioned in a temperature-controlled water bath or environmental chamber to maintain the target test temperature. Each test chamber will be labeled with the project number, test concentration and replicate designation.
- Type of flow-through: A continuous-flow diluter system will be used to provide each concentration of the test substance, a negative (dilution water) control, and when necessary, a solvent control. One or more pumps (e.g., syringe pump, peristaltic pump, or metering pump) or a similar device will be used to deliver test substance stock solutions to mixing chambers where the test substance will be mixed with dilution water to prepare the test solutions at the appropriate nominal concentrations prior to delivery to the test chambers. In tests where a solvent control is required, the solvent will be delivered to a separate mixing chamber where it will be diluted to the appropriate concentration with dilution water. The flow of dilution water into each mixing chamber will be controlled using rotameters. After mixing, test solutions will be delivered in approximately equal volumes to each replicate test chamber within an exposure group. The proportion of water delivered to each replicate will be checked prior to exposure initiation and near the end of the test (e.g., one day prior to termination), and approximately weekly or as needed during the test to ensure that these flow rates vary by no more than ±10% of the mean flow rate of the four replicates.
The diluter will be adjusted so that each test chamber receives at least 5 volume additions of test solution every 24 hours. Peristaltic or metering pumps, if used, and rotameters will be calibrated prior to exposure initiation and verified near the end of the test (e.g., one day prior to termination), and recalibrated and/or verified approximately weekly or as needed during the test. Syringe pumps, if used, will be calibrated prior to exposure initiation, and verified near the end of the test (e.g., one day prior to termination) and as needed during the test. The delivery of test substance to test chambers will begin at least one day prior to exposure initiation in order to establish equilibrium concentrations of the test substance. The duration of the equilibration period typically will be dependent on the physical/chemical properties of the test substance. The general operation of the diluter system will be checked visually at least two times per day during the test and at least once on the first and last days of the test. The delivery system and chambers will be cleaned periodically during the test, as needed.
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used for organism holding and testing will be obtained from a well approximately 40 meters deep located at the test facility. The water will be passed through a sand filter and pumped into a 37,800-L storage tank where the water will be aerated with spray nozzles. Prior to use in the test system, the water will be filtered to 0.45 µm in order to remove fine particles and will be UV-sterilized. The water is characterized as moderately hard.
- Hardness dilution water: 140 mg/L as CaCO3
- Alkalinity dilution water: 180 mg/L as CaCO3
- pH dilution water: 8.2
- Specific conductance dilution water: 365 µS/cm
- Total organic carbon dilution water: <2 mg/L
- Solubility of the test material: Observations of the test solution appearance in the diluter system mixing chambers and in the test chambers will be recorded at the beginning and end of the test, and as needed during the test if the test solution appearance changes.

OTHER TEST CONDITIONS
- Light quality: The test systems will be illuminated using fluorescent tubes that emit wavelengths similar to natural sunlight.
- Photoperiod: The lights will be controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity will be provided when lights go on and off to avoid sudden changes in light intensity.
- Light intensity: The target light intensity will be in the range of approximately 540 to 1080 lux. Light intensity at the water surface of at least one representative test chamber will be measured at the beginning of the test with a SPER Scientific Ltd. light meter or equivalent.

WATER QUALITY MEASUREMENTS
- Temperature: The test will be conducted at a target water temperature of 20 ± 1°C. Temperature will be measured in each test chamber at the beginning of the test, approximately weekly during the test, and at the end of the test using a liquid-in-glass or digital thermometer, or equivalent. Water temperature also will be monitored continuously during the test in at least one representative vessel using an automatic monitoring system. The system measurements will be verified with a liquid-in-glass or digital thermometer, or equivalent, prior to exposure initiation and approximately weekly or as needed during the test.
- Dissolved oxygen: Dissolved oxygen will be measured in one replicate test chamber of each treatment and control group at the beginning of the test, approximately three times per week during the test (e.g., Monday, Wednesday and Friday), and at the end of the test. Measurements typically will rotate among the replicates in each group at each measurement interval. The dilution water is aerated prior to use so that dissolved oxygen concentrations typically are at or near 90 to 100% saturation at the beginning of the test. In the event that dissolved oxygen levels approach or fall below 60% saturation during the test, appropriate corrective actions (e.g., increased flow rates, increased cleaning frequency, or aeration of the test solutions as a last resort) will be taken, if necessary, to maintain dissolved oxygen levels above 60% saturation. Dissolved oxygen measurements will be made using a Thermo Orion Star A213 dissolved oxygen meter, or equivalent.
- pH: Water pH will be measured in one replicate test chamber of each treatment and control group at the beginning of the test, approximately weekly during the test, and at the end of the test. Measurements typically will rotate among the replicates in each group at each measurement interval. The pH should be in the range of 6.0 to 8.5 and should not vary more than 1.0 pH unit during the test. Measurements of pH will be made using a Thermo Orion Dual Star pH/ISE meter, or equivalent.
- Hardness, pH, alkalinity: Hardness, alkalinity and specific conductance will be measured in one replicate test chamber of the negative (dilution water) control and the highest concentration treatment group at the beginning of the test, approximately weekly during the test, and at the end of the test. Measurements typically will rotate among the replicates in each group at each measurement interval. Hardness and alkalinity measurements will be made by titration based on methods in Standard Methods for the Examination of Water and Wastewater. Specific conductance will be measured using a Thermo Orion Star A122 conductivity meter, or equivalent.

EFFECT PARAMETERS MEASURED:
The parental-generation daphnids will be observed daily during the test for survival/immobility, clinical signs of toxicity, abnormal behavior or appearance, and the onset of reproduction (time in days to the release of the first brood in each test vessel). Those daphnids that are not able to swim within 15 seconds after gentle agitation of the test vessel are considered to be immobilized (even if they can still move their antennae). Immobilization is used as a surrogate for death. Therefore, dead animals will be counted as immobile. Any immobile parental generation daphnids will be removed from the test compartments at each observation period. Examples of clinical signs of toxicity include inability to maintain position in the water column, discoloration (e.g., pale or dark) and cessation of feeding. The presence of eggs in the brood pouch, and the numbers of males or ephippia, if any, will be recorded daily.

With the onset of reproduction, the numbers of young (F1 generation) daphnids will be counted in each test compartment approximately three times per week (e.g., Monday, Wednesday and Friday). The number of aborted eggs, if any, also will be recorded at each counting interval. Live F0-generation daphnids will be retained and F1-generation daphnids will be discarded. At the end of the test, the length of each surviving F0-generation daphnid will be measured to the nearest 0.1 mm using digital calipers or equivalent, and dry weight will then be measured to the nearest 0.01 mg after drying the organisms at approximately 60 ºC for at least 24 hours. Throughout the exposure period, the test organisms periodically may be transferred to clean test compartments and/or test chambers, as necessary.

Reference substance (positive control):

no

Remarks on result:

other: Study planned

Description of key information

As a result of the aquatic toxicity data information submitted, it occurred that chronic effects cannot be excluded for the substance (being poorly water soluble and not showing acute effects). In this case, long-term testing shall be considered in accordance with Regulation (EC) No 1907/2006 (REACH). Even though there already is long-term fish data available, chronic daphnid data should still be generated since there is no indication that daphnids are the least sensitive species. A long-term toxicity study to aquatic invertebrates is currently ongoing and due to some methodological difficulties in validating the analytical methods, it is expected to be finalised in November 2019. As soon as the information is available, the risk assessment and CSR will be updated.