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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:
12 Jul 2018 to 14 Jul 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)
Version / remarks:
2004
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
2016
Qualifier:
according to guideline
Guideline:
other: ASTM Standard E 729 96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
Version / remarks:
2014
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Perkacit® TDEC pdr
- Batch No.: 60701019
- Appearance: Solid
- Tellurium content: 17.6%
- Date of manufacture: 09 Feb 2017
- Storage conditions: Ambient
Analytical monitoring:
yes
Details on sampling:
Test water samples were collected from alternating test chambers of each treatment and control group three and one days prior to the start of exposure to confirm concentrations after conditioning the diluter system for four and six days, respectively. Test water samples also were collected from one replicate test chamber in each treatment and control group 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. The samples (10.0 mL) were collected from mid-depth, placed in glass vials containing 10.0 mL of acetonitrile and processed immediately for analysis.
Vehicle:
yes
Remarks:
100 µL/L dimethylformamide (DMF; HPLC-grade)
Details on test solutions:
Individual stock solutions were prepared for each of the five concentrations tested and were prepared daily during the test. 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 300 or 50-mL primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 1600 µg/mL. The primary stock solution was partially brought to volume and sonicated for approximately five or 15 minutes. The stock solution was then brought to final volume and stirred on a magnetic stir plate for approximately 10 or 15 minutes. The stock solution appeared clear and orange or golden yellow with no visible precipitates. Four secondary stock solutions (25 mL each) were prepared in DMF at nominal concentrations of 26, 73, 200 and 570 µg/mL by proportional dilution of the primary stock. The secondary stock solutions were mixed by stirring on a magnetic stir plate for approximately 15 minutes and ranged in appearance from clear and colorless to clear and yellow or orange, with no evidence of precipitates.

The stock solutions were held at room temperature. Fresh aliquots of each stock were placed on the delivery system pumps every day 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.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnids
- Source: Cultures maintained by the test facility
- Age: Daphnids used in the test were <24-hour old neonates
- Feeding during test: No

HOLDING
- Parental stock: The five adult daphnids used to supply neonates for the test were held for 21 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 0% in the two-day period prior to test initiation.
- Acclimation conditions: During the 2 week period immediately preceding the test, water temperatures in the cultures ranged from 19.7 to 20.5ºC, the pH of the water ranged from 8.0 to 8.4, and the dissolved oxygen concentrations were ≥7.4 mg/L (≥81% of saturation).
- Feeding: 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:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
144 mg/L as CaCO3 (at Day 0)
Test temperature:
Manual measurements: 20.1 - 20.3 °C. Temperature monitored continuously during the test ranged from 19.99 to 20.11°C, measured to the nearest 0.01°C.
pH:
8.0 - 8.4
Dissolved oxygen:
7.8 - 8.9 mg O2/L
Conductivity:
335 µS/cm (at Day 0)
Nominal and measured concentrations:
- Nominal concentrations: 0 (blank control), 0 (solvent control), 2.6, 7.3, 20, 57 and 160 µg/L.
- Mean measured concentrations:
Details on test conditions:
TEST SYSTEM
- Test vessel: Test chambers were 25 L Teflon®-lined stainless steel aquaria. The depth of the test water in a representative chamber was 30.0 cm and was maintained by a standpipe within the test chamber. Each test chamber contained two test compartments constructed from a glass beaker 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. The test chambers were impartially positioned in a temperature-controlled water bath to maintain the target water temperature throughout the test period.
- Fill volume: 22 L of test water.
- Aeration: No
- 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 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. Impellors were located in the mixing chambers to facilitate mixing of the stock solutions with the dilution water. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately five 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 exposure initiation and verified at the end of the test. The proportion of the test water that was delivered to each replicate test chamber was checked 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 seven 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: 10 (5 per compartment)
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2

TEST MEDIUM / WATER PARAMETERS
- 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 EAG Laboratories site in Easton, Maryland. 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.
- Total organic carbon: <1 mg C/L in the dilution water
- Alkalinity: 180 mg/L as CaCO3

OTHER TEST CONDITIONS
- Adjustment of pH: No
- 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 quality: The test systems were illuminated using fluorescent tubes that emit wavelengths similar to natural sunlight.
- Light intensity: Light intensity at the beginning of the test was 577 lux at the surface of the water of one representative test chamber. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006 light meter.

WATER QUALITY MEASUREMENTS
- Temperature: The test was conducted at a target water temperature of 20 ± 1 °C. Temperature was measured in each replicate test chamber of each treatment and control group at the beginning of the test, at approximately 24 hours during the test, and at the end of the test using a digital thermometer. Water temperature also was monitored continuously in one negative control test chamber using a validated environmental monitoring system (AmegaView Central Monitoring System). The system measurements were calibrated prior to exposure initiation with a digital thermometer.
- Dissolved oxygen and pH: Dissolved oxygen and pH were measured in each replicate test chamber of each treatment and control group at the beginning of the test, at approximately 24 hours during the test, and at the end of the test. Dissolved oxygen was measured using a Thermo Scientific Orion Star A213 Benchtop RDO/DO meter, and measurements of pH were made using a Thermo Scientific Orion DUAL STAR pH/ISE meter.
- Hardness, akalinity, specific conductance: Hardness, alkalinity and specific conductance in the dilution water at the beginning of the test were measured. Hardness and alkalinity measurements were made by titration based on methods in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using a Thermo Scientific Orion Star A122 portable conductivity meter.

EFFECT PARAMETERS MEASURED: immobility, sub-lethal effects
Prior to test initiation, neonate daphnids < 24 hours old were collected from the cultures and were impartially distributed one or two at a time to transfer containers until each contained its complement of five daphnids. To initiate the test, the transfer containers were impartially assigned to test compartments, and the daphnids were released into the test compartments. 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.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
The range-finding study was conducted at nominal concentrations of 2.5, 9.9, 40, 159 and 634 µg/L for 48 hours under flow-through test conditions. Percent immobility in the 2.5, 9.9, 40, 159 and 634 µg/L treatment groups at test termination was 0, 70, 60, 90 and 100%, respectively. Sublethal effects included lethargy and floating at test termination. Based on these findings, the concnetrations for the definitive test were determined.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
64 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.I.: 37 - 141 µg/L
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 136 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Test substance appearance: The test solutions in the test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution. The solution in the diluter mixing chambers that delivered test solutions for the 2.6 and 7.3 µg/L test concentrations also appeared clear and colorless, with no evidence of precipitation. The solution in the diluter mixing chambers that delivered test solutions for the 20, 57 and 160 µg/L test concentrations also appeared clear and colorless, but yellow precipitate was noted on the sides of the mixing chamber, siphon cap, impellor and pipette delivering the dilution water.
- Observations controls: All daphnids in the negative and solvent control groups appeared normal throughout the test.
- Observations treatment groups: All daphnids in lowest two treatment groups also appeared normal throughout the test, with no immobile daphnids or overt signs of toxicity observed. There was one floating daphnid at 24 hours in the lowest treatment group, but it appeared normal after gentle submersion. Percent immobility in the 1.1, 1.1, 16, 50 and 136 µg/L treatment groups at test termination was 0, 0, 25, 50 and 60%, respectively. Signs of toxicity observed among the surviving daphnids in the 16, 50 and 136 µg/L treatment groups at test termination included lethargy.
Reported statistics and error estimates:
The immobility data were analyzed using the computer program of C. E. Stephan. The program was designed to calculate the EC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation. Based on the immobility pattern in this study, probit analysis was used to calculate the 48 hour EC50 value. Since there was <50% immobility at 24 hours, the 24 hour EC50 value was estimated to be greater than the highest concentration tested. The no-immobility concentration and the 100% immobility concentration were determined by visual interpretation of the immobility data. The no-observed-effect concentration (NOEC) based on immobility was analyzed using Cochran-Armitage (O) Trend Step-Down Test in CETIS™ v1.9.3.0

Table: Cumulative immobility and observations

Nominal

Concentration

(µg/L)

 

 

Rep.

 

No.

Exposed

~4 Hours

24 Hours

48 Hours

Cumulative

Percent

Immobility

Number

Immobile (1)

 

Observations (2)

Number

Immobile (1)

 

Observations (2)

Number

Immobile (1)1

 

Observations (2)

Negative Control

A

10

0

10 AN

0

10 AN

0

10 AN

0

B

10

0

10 AN

0

10 AN

0

10 AN

Solvent Control

A

10

0

10 AN

0

10 AN

0

10 AN

0

B

10

0

10 AN

0

10 AN

0

10 AN

2.6

A

10

0

10 AN

0

10 AN

0

10 AN

0

B

10

0

10 AN

0

1 Q,AN; 9 AN

0

10 AN

7.3

A

10

0

10 AN

0

10 AN

0

10 AN

0

B

10

0

10 AN

0

10 AN

0

10 AN

20

A

10

0

1 C; 9 AN

0

1 C; 4 Q,AN; 5 AN

2

5 C; 3 AN

25

B

10

0

1 Q,AN; 9 AN

0

5 Q,AN; 5 AN

3

3 C; 4 AN

57

A

10

0

10 AN

0

7 Q,AN; 3 AN

4

5 C; 1 AN

50

B

10

0

1 Q,AN; 9 AN

0

5 C; 5 AN

6

3 C; 1 AN

160

A

10

0

6 Q,AN; 4 AN

0

5 Q,AN; 5 AN

5

5 C

60

B

10

0

6 Q,AN; 4 AN

0

2 Q,C; 7 Q,AN; 1 AN

7

3 C

Observations of surviving organisms: AN = appear normal; C = lethargy; Q,AN = trapped at water surface but appear normal after gentle submersion; Q,C = trapped at water surface and appear lethargic after gentle submersion.

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.
Executive summary:

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria (EAG Inc., 2018). In this study daphnids (D. magna, 20 per concentration) were exposed to nominal concentrations of 0 (blank control), 0 (solvent control), 2.6, 7.3, 20, 57 and 160 µg/L for 48 hours under flow-though conditions. Analytically verified test concentrations did not remain within ± 20% of the nominal concentrations. Therefore, the mean measured concentrations of <LOQ (blank control), LOQ (solvent control), 1.1, 1.1, 16, 50 and 136 µg/L (arithmic mean) were used for the derivation of the effect values. Immobilization was recorded after ~4, 24 and 48 hours exposure. All daphnids in the negative and solvent control groups appeared normal throughout the test. All daphnids in lowest two treatment groups also appeared normal throughout the test, with no immobile daphnids or overt signs of toxicity observed. There was one floating daphnid at 24 hours in the lowest treatment group, but it appeared normal after gentle submersion. Percent immobility in the 1.1, 1.1, 16, 50 and 136 µg/L treatment groups at test termination was 0, 0, 25, 50 and 60%, respectively.  Signs of toxicity observed among the surviving daphnids in the 16, 50 and 136 µg/L treatment groups at test termination included lethargy. Based on these findings the 48-h EC50 value was determined at 64 µg/L.

Description of key information

The 48-h EC50 was determined to be 64 μg/L in freshwater invertebrates Daphnia magna, according to OECD TG 202 and GLP criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
64 µg/L

Additional information