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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
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
Analytical monitoring:
yes
Details on sampling:
At the start of the test, the excess test solutions were sampled, and at the end of the test one replicate of the dilution water and solvent control and each test concentration was sampled.
Vehicle:
yes
Remarks:
Dimethylformamide (DMF)
Details on test solutions:
A primary stock solution was prepared by dissolving 0.1 g of test substance in 100 mL of an organic solvent, DMF. The stock solution was a pale yellow solution.
A further intermediate stock solution was prepared by diluting 10 mL of the 1.0 g/L stock solution to 100 mL with DMF. The intermediate stock solution was clear and colourless.
One litre of each test solution was prepared by the addition of aliquots of the stock solution to dilution water. The additions were made gradually by microlitre syringe while stirring with a magnetic follower. Equalizing additions of DMF were made in the same way, such that all test concentrations and the solvent control contained 0.1 mL of DMF per litre. The control consisted of dilution water only.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Waterflea
- Strain/clone: Planktonic crustacean
- Source: Brixham Environmental Laboratory
- Age of parental stock: 18 ± 1 days
- Feeding during test: Not fed
- Food type: Algae Chlorella vulgaris (microencapsulated diet)

STOCK CULTURES:
- Temperature: 20 ± 2°C
- Photoperiod: 16 hours light : 8 hours dark (with 20 minute transition periods)
- Culture condition: Reproduction was by diploid parthenogenesis
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20 ± 1°C
pH:
8.11 to 8.15
Dissolved oxygen:
9.0 to 9.2 mg/L
Nominal and measured concentrations:
Nominal: Dilution water control, solvent control, 3.2, 5.6, 10, 18, 32, 56, 100 µg/L
Measured: <0.19 (dilution water and solvent controls), 3.2, 5.7, 10, 19, 32, 58, 99 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Borosilicate glass beakers (250 mL). Each vessel contained 200 mL of test solution providing a depth of approximately 60 mm
- Type: Closed
- Aeration: No aeration
- No. of organisms per vessel: 20
- Loading of organisms: 25 Daphnia/L
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Medium: Elendt's M4 Daphnia medium
- Alkalinity as CaCO3: 49 mg/L
- Conductivity: 663 µS/cm
- Hardness (total as CaCO3): 248 mg/L
- pH: 8.1
- Non purgeable organic carbon: 1.7 mg/L

OTHER TEST CONDITIONS
- Test solution temperature: 20 ± 2°C
- Photoperiod: 16 hours light : 8 hours dark (with 20 minute transition periods)

EFFECT PARAMETERS MEASURED: Assessments of the response of the Daphnia were made at 24 and 48 hours after commencement of the test. Each Daphnia was viewed by eye, and was defined as affected if showing no whole body movement relative to the water within a period of 15 seconds, even if movement of individual appendages was visible. Daphnia so affected were termed immobile.

VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
24 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Immobility
Remarks on result:
other: 95% CI: 18-32 µg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
18 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Immobility
Validity criteria fulfilled:
yes
Conclusions:
48 h EC50: 24 µg/L (95% CI: 18-32 µg/L) (Immobility)
48 h NOEC: 18 µg/L (Immobility)
Executive summary:

A study on the acute toxicity of the test substance to Daphnia magna was conducted according to OECD guideline 202.

The static bioassay procedure was followed to determined 48 hours EC50 value. Daphnia were exposed to dilution water control, solvent control and nominal concentrations of 3.2, 5.6, 10, 18, 32, 56, and 100 µg/L. Daphnia were observed for immobility at 24 and 48 hours after commencement of the test.

The lowest nominal concentration at which there was 100% immobilization at 48 hours was 32 µg/L. The 48 hour EC50 based on immobility was 24 µg/L and NOEC was 18 µg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1025 (Bivalve Acute Toxicity (shell deposition test))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: U.S. EPA OPPTS 850.1000 (Special Consideration for Conducting Aquatic Laboratory Studies, "Public Draft", EPA 712-C-96-113)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: U.S. EPA-540/9-85-011 (Mollusc 96-Hour Flow-Through Shell Deposition Study)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
One replicate of the high, middle and low treatment levels and the dilution water control were sampled prior to the start of the definitive exposure and analyzed for test substance active ingredient. In addition, a sample of the diluter stock solution was analyzed for test substance active ingredient. Results of these pretest analyses were used to judge whether sufficient quantities of test substance were being delivered and maintained in the exposure aquaria to initiate the oyster shell deposition exposure.

During the definitive study, samples were removed from one replicate solution of each treatment level and the controls at each sampling interval. At test initiation, replicate A was sampled and at test termination, replicate B was sampled and analyzed for test substance active ingredient. In addition, a sample of the 0.32 mg a.i./mL stock solution was also analyzed for picoxystrobin a.i. at the 0- and 96-hour sampling intervals.

Three quality control (QC) samples were prepared at each sampling interval and remained with the exposure solution samples throughout the analytical process.
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
Diluter Stock Solution Preparation: A 0.32 mg a.i./mL primary stock solution was prepared by adding 0.0645 g of test substance (0.0640 g as a.i.) to a 200-mL volumetric flask and bringing it to volume with dimethylformamide (DMF). The resulting stock solution was observed to be clear and colorless.
Test organisms (species):
other aquatic mollusc: Crassostrea virginica
Details on test organisms:
TEST ORGANISM
- Common name: Eastern oysters
- Justification for species other than prescribed by test guideline: Eastern oysters were selected because of the availability of the species
- Source: Circle C Oysters, Ridge, Maryland
- Age at dosing: Juveniles
- Length at dosing: 33 to 46 mm
- Food type: Supplementary algal diet of Tetraselmis maculata prepared in seawater from a concentrate
- Amount/frequency: Concentrated volumes of algal suspension (approximately 107 cells/mL) were added to each test aquarium three times daily to maintain an average concentration of approximately 105 cells/mL in the test solutions during the four-day exposure.

ACCLIMATION
- Acclimation period: 28 days
- Acclimation conditions: During this acclimation period, the seawater in which the oysters were held had a temperature range of 15 to 20ºC, a pH range of 7.3 to 7.9 and a dissolved oxygen concentration range of 74 to 100% of saturation. Salinity was maintained at 20 to 21%
- Health during acclimation: No mortality observed

SELECTION:
Upon arrival, the oysters were carefully examined. If any oyster appeared unsuitable for testing due to the presence of boring sponges and/or mudworms, it was discarded. In addition, several oysters were opened and carefully examined to confirm that no parasites were present, then discarded. These oysters were also determined to be reproductively immature by pressing the tissue in the area where gametes are stored to confirm that none were present. The oysters were of similar age and had a mean valve height of 38 ± 4 mm (N = 30).
Test type:
flow-through
Water media type:
saltwater
Limit test:
yes
Total exposure duration:
96 h
Test temperature:
19 to 22ºC
pH:
7.5 to 7.9
Dissolved oxygen:
6.1 to 8.0 mg/L
Salinity:
20 to 21‰
Nominal and measured concentrations:
Nominal: 1.5, 2.7, 4.8, 8.8 and 16 μg a.i./L
Measured: 1.4, 2.3, 4.5, 7.7, and 12 μg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria
- Material, size, headspace, fill volume: 49.5 x 25.5 x 29 cm, 18 L
- Flow: 75 mL/minute
- No. of organisms per vessel: 20 (40 per treatment level and the controls)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light:8 hours dark
- Light intensity: 540 to 1300 lux

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
- Test concentrations: 1.9, 4.9, 13, 34, and 89 μg a.i./L
- Results used to determine the conditions for the definitive study: Statistical analysis (Williams’ Test) determined a significant reduction in shell growth among oysters exposed to all treatment levels tested compared to the blank control. No mortality or adverse effects were observed among oysters exposed to any of the concentrations tested or the blank control. The preliminary range-finding test with this test substance was conducted without the use of a co-solvent. Analytical sampling performed in conjunction with this exposure resulted in recoveries of approximately 20% of nominal concentrations. Additional solubility trials with the test substance were conducted to evaluate the use of a co-solvent (i.e., dimethylformamide, DMF). Based on these results, DMF was used to solubilize the test material for the definitive testing and the nominal concentrations selected for the definitive test were 1.5, 2.7, 4.8, 8.8 and 16 μg a.i./L.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
5.7 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: reduction in shell growth
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: significant reduction in shell growth
Reported statistics and error estimates:
Statistical analysis determined no significant difference between blank control and solvent control data, therefore negative control data was used for further statistical analysis. No mortality or adverse effects were observed among oysters at any of the treatment levels tested or the controls. Following 96 hours of exposure, reduction in shell growth among oysters exposed to the 1.4, 2.3, 4.5, 7.7, and 12 μg a.i./L mean, measured test concentrations was 19, 28, 42, 69 and 63%, respectively. Statistical analysis (Williams' Test) determined a significant difference in mean, measured test concentrations ≥ 2.3 μg a.i./L when compared to the control. Therefore, the No-Observed-Effect Concentration (NOEC) was determined to be 1.4 μg a.i./L based on mean, measured test concentrations and the significant reduction in shell growth. The 96-hour EC50 based on mean, measured test concentrations and the reduction in shell growth was calculated by linear regression to be 5.7 μg a.i./L, with corresponding 95% confidence limits of 1.7 to 25 μg a.i./L.
Validity criteria fulfilled:
yes
Conclusions:
96 h EC50: 5.7 μg a.i./L (reduction in shell growth)
96 h NOEC: 1.4 μg a.i./L (significant reduction in shell growth)
Executive summary:

The acute toxicity of the test substance to Eastern oyster, Crassostrea virginica, under flow-through conditions was determined in a 96-hour exposure according to EPA OPPTS 850.1025 and 850.1000 guidelines.

The study was conducted with five concentrations of test substance, a dilution water control and a solvent (dimethylformamide, DMF) control at a temperature range of 19 to 22°C. Two replicates with 20 oysters per replicate were initiated for each test substance concentration and control.

Exposure of oysters to mean, measured test substance concentrations of 1.4, 2.3, 4.5, 7.7, and 12 μg a.i./L resulted in mean percent reduction of shell growth of 19, 28, 42, 69, and 63, respectively, at the end of 96 hours. Mean, measured concentrations ranged from 76 to 96% of the nominal concentrations. The highest mean, measured concentration causing no statistically significant reduction (NOEC) in growth was 1.4 μg a.i./L. Mean, measured concentrations of test substance were used for calculation of EC50 and NOEC values. The 96-hour EC50, based on mean, measured concentrations of picoxystrobin and the reduction in shell growth was 5.7 μg a.i./L.

Description of key information

48-hour EC50 (Daphnia magna): 0.024 mg/L; OECD 201; Reliability = 1

96-hour EC50 (Crassostrea virginicia): 0.0057 mg/L; EPA OPPTS 850.1025, EPA OPPTS 850.1000, EPA-540/9 -85 -011; Reliability = 1

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.024 mg/L

Marine water invertebrates

Marine water invertebrates
Effect concentration:
0.006 mg/L

Additional information

Acute aquatic invertebrate tests were conducted in one freshwater species and two marine species. The 48-h EC50 value in Daphnia magna is 0.024 mg a.s./L, based on nominal test concentrations. Marine species tested Crassostrea virginica and Americamysis bahia observed 96-h EC50/LC50 values, based on mean measured concentrations, of 0.0057 and 0.033 mg a.s./L, respectively. The most sensitive acute 48h EC50 for freshwater aquatic invertebrates is less than 1 mg a.s./L.