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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Apr 2018 to 8 Apr 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
according to guideline
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
GLP compliance:
Specific details on test material used for the study:
- Name of test material (as cited in study report): Nickel Dibutyldithiocarbamate (NDBC)
- Date of receipt: October 3, 2017
- Storage conditions: The material was stored under ambient conditions
- Appearance: solid
- Lot No.: 70900202
Analytical monitoring:
Details on sampling:
Test water samples were collected from alternating test chambers of the 2.5, 5.0 and 10 μg/L treatment groups and both control groups 10, 4, 3 and 1 days prior to the start of exposure to confirm concentrations after conditioning the diluter system for 1, 7, 8 and 10 days, respectively. Duplicate test water samples also were collected from one replicate test chamber of the 2.5, 5.0 and 10 μg/L treatment groups and both control groups at the beginning of the test and at 48 hours (± 1 hour) to measure concentrations of the test substance. Sampling alternated between the replicate test chambers in each group at each sampling interval starting 4 days prior to the start of the exposure. Samples (30 mL) were collected from mid-depth and placed in Teflon® centrifuge tubes. The Teflon® centrifuge tubes were preconditioned by filling with the appropriate test solution and allowing to sit overnight for all sampling intervals except 10 days prior to the start of the exposure. One set of samples collected at each sampling interval was processed immediately for analysis. The duplicate set of samples was stored under refrigeration as back-up samples for possible analysis. Stock solution samples were collected from the 6.3 and 13 μg/L stock solutions 10, 4, 3 and 1 days prior to the start of exposure to confirm concentrations after conditioning the diluter system for 1, 7, 8 and 10 days, respectively. Stock solution samples were also collected from the 6.3 and 13 μg/L stock solutions at the beginning of the test and at 48 hours (± 1 hour) to measure concentrations of the test substance in the stock solutions used to prepare the 6.3 and 13 μg/L treatment groups. Samples (3 mL) were collected from the syringe and placed in glass vials and processed immediately for analysis.
Details on test solutions:
Individual stock solutions were prepared for each of the five concentrations tested using preconditioned glassware and were prepared four times during the test. The glassware was preconditioned by preparing test solutions and letting them sit in the glassware overnight. The glassware was reused for subsequent stock preparations. Test solution concentrations were not adjusted for the active ingredient of the test substance during preparation, and are based on the test substance as received. A 200-mL primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 100 μg/mL. The primary stock solution was brought to partial volume and sonicated for approximately 10 minutes. It was then brought to final volume and stirred on a magnetic stir plate for approximately 10 minutes, and appeared clear and green or light green with no visible precipitates. Four secondary stock solutions (100 mL each) were prepared in DMF at nominal concentrations of 6.3, 13, 25 and 50 μg/mL by proportional dilution of the primary stock. The secondary stock solutions were mixed by stirring on a magnetic stir plate for approximately 10 minutes, and appeared clear and increasingly green with an increase in concentration. The stock solutions were held under refrigerated conditions in glass amber bottles. Fresh aliquots of each stock were placed on the delivery system pumps every one or two days during the test. During the exposure period, the stock solutions were pumped into the diluter mixing chambers assigned to the treatment groups at a target rate of 15.5 μL/minute and were mixed with dilution water in the mixing chambers, delivered at a target rate of 155 mL/minute to achieve the desired nominal test concentrations. The negative control received dilution water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control at the same rate as the test substance stock solutions. The concentration of DMF in the solvent control and all treatment groups was 100 μL/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Source: Test facility
- Age: < 24h-old neonates

Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.6 to 20.5 ºC, the pH of the water ranged from 8.1 to 8.4, and the dissolved oxygen concentrations were ≥7.6 mg/L (≥84% of saturation). The four adult daphnids used to supply neonates for the test were held for 24 days prior to collection of the juveniles for testing, and had each produced at least four previous broods. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress, no ephippia were produced during the holding period, and mortality in the culture stock was <10% in the two-day period prior to test initiation.

Daphnids in the cultures were fed daily 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. The adults were fed during the 24-hour period prior to test initiation, but neonates were not fed during the test.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
136 mg/L as CaCO3 at t=0
Test temperature:
19.6 - 20.3 °C manual measurements. Temperature monitored continuously during the test ranged from 19.37 to 20.18°C, measured to the nearest 0.01 °C.
7.9 - 8.1
Dissolved oxygen:
7.0 - 8.9
322 µS/cm at t=0
Nominal and measured concentrations:
- Nominal concentrations: 0 (negative control), 0 (solvent control), 0.63, 1.3, 2.5, 5.0 and 10.0 µg/L
- Mean measured concentrations:
Details on test conditions:
- Test vessel: Test chambers were 25-L Teflon®-lined stainless steel aquaria filled with 22 L of test water. The depth of the test water in a representative chamber was 29.8 cm, and was maintained by a standpipe within the test chamber. Each test chamber contained two test compartments constructed from a glass beaker approximately 6.5 cm in diameter and 12 cm in height, with two nylon mesh-covered holes on opposite sides of the container to allow for the flow of water through the test compartment. The depth of the test water in a representative compartment was 10.0 cm. All test chambers were labeled with the project number, test concentration and replicate designation. The test chambers were impartially positioned in a temperature-controlled water bath to maintain the target water temperature throughout the test period.
- Type of flow-through: The toxicity test was conducted using a continuous-flow diluter system to provide each concentration of the test substance, a negative control (dilution water only) and a solvent control (100 μL dimethylformamide/L) to test chambers. Syringe pumps were used to deliver volumes of test substance stock solutions to mixing chambers impartially assigned to each treatment group. Dimethylformamide (DMF) was delivered to a separate mixing chamber assigned to the solvent control. The stock solutions or solvent were mixed with well water in the mixing chambers in order to prepare the test solutions at the appropriate nominal concentrations prior to delivery to the test chambers. Well water alone was delivered to a mixing chamber for the negative control. The flow of
dilution water into each mixing chamber was controlled using rotameters, and was adjusted to provide approximately 5 volume additions of test solution in each test chamber per day. After mixing, the test water in each mixing chamber was delivered in approximately equal volumes to two replicate test chambers in each control and treatment group. The pumps used to deliver stock solutions or solvent to the mixing chambers, and the rotameters used to control the flow of dilution water to the mixing chambers, were calibrated prior to the test and at the end of the test. The proportion of the test water that was delivered to each replicate test chamber was checked prior to the test and at the end of the test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the two replicates. Delivery of test water to the test chambers was initiated 11 days prior to the introduction of the test organisms to the test water in order to establish equilibrium concentrations of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and at least two times per day during the test.
- No. of organisms per vessel: 5
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2

- Source/preparation of dilution water: The water used for organism holding and testing was freshwater obtained from a well approximately 40 meters deep located on the test facility. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use in the test system, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The well water is characterized as moderately-hard water.
- Alkalinity: 180 mg/L as CaCO3 at t=0

- Photoperiod: The lights were 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 was provided when lights went on and off to avoid sudden changes in light intensity.
- Light intensity: Light intensity at the beginning of the test was 969 lux at the surface of the water of one representative test chamber.

EFFECT PARAMETERS MEASURED: immobility and sub-lethal effects
Prior to test initiation, neonate daphnids < 24 hours old were collected from the cultures and were impartially distributed one and two at a time to transfer containers until each contained its complement of 5 daphnids. To initiate the test, the transfer containers were impartially assigned to test compartments, and the daphnids were released into the test compartments one or two at a time. All daphnid transfers were conducted below the water surface using wide-bore pipettes. All organisms were observed periodically during the test to determine the number of immobile organisms in each treatment group. Those daphnids that were not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobilized (even if they could still move their antennae). The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4, 24 and 48 hours after test initiation.
Reference substance (positive control):
Key result
48 h
Dose descriptor:
Effect conc.:
> 6.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
All daphnids in the negative and solvent control groups appeared normal throughout the test. All daphnids in the 0.63, 1.3, 2.5, 5.0 and 10 μg/L treatment groups also appeared normal throughout the test, with no immobile daphnids or overt signs of toxicity observed. Percent immobility in the 0.63, 1.3, 2.5, 5.0 and 10 μg/L treatment groups at test termination was 0, 0, 0, 0 and 0%, respectively. No sublethal effects were observed among the surviving daphnids in any treatment groups at test termination.
Reported statistics and error estimates:
The absence of immobility in any of the treatment groups during the test precluded the statistical calculation of EC50 values at 24 and 48 hours. Therefore, the EC50 values were estimated to be greater than the highest concentration tested. The no-immobility concentration and the no-observed-effect concentration (NOEC) were empirically estimated from the immobility data.
Validity criteria fulfilled:
See 'Any other information on materials and methods incl. tables'

Description of key information

48-h EC50 > 6.3 µg/L (Daphnia magna)

Key value for chemical safety assessment

Additional information

The toxicity towards freshwater invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria (EAG, 2018). In this study, juvenile Daphnia magna were exposed to solutions of the test substance of 0, (control), 0 (solvent control), 0.63, 1.3, 2.5, 5.0, 10 μg/L (nominal concentrations) for 48 hours under flow-through conditions. The test was performed with two replicates per test concentration, each holding 5 daphnids. The initial concentrations and the maintenance of the exposure concentrations during the test were analytically determined. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations of the three highest treatments for this study were 1.8, 3.5 and 6.3 μg/L representing 72, 70 and 63% of nominal concentrations, respectively. The results of the study were therefore based on the mean measured concentrations. Daphnids in all treatments appeared normal throughout the test with no immobile daphnids or overt signs of toxicity observed. The 48-h EC50 was therefore determined to be > 6.3 µg/L.