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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Apr 2014 - 25 Apr 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, solvent control, 5.00 and 10.00 mg a.i./L
- Sampling method: Water samples were taken daily between day 0 (test initiation) and day 4 (test termination)
Vehicle:
yes
Remarks:
dimethylformamide (DMF)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions were prepared daily by adding the appropriate amounts of stock solution to synthetic seawater. Stock solutions prepared on study day 0 were utilized to spike new test solutions daily.
- Controls: control, solvent control
- Chemical name of vehicle: DMF
- Concentration of vehicle in test medium: 100 μL/L
- Evidence of undissolved material: Precipitates were observed in the test level with 10.0 mg a.i./L in aged test solutions, no precipitates were observed in the test level with 5.00 mg a.i./L.
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISM
- Common name: Sheepshead Minnow
- Culture Lot No.: ABS040214
- Source: Aquatic Bio Systems, Fort Collins, CO, United States
- Age at study initiation: Juvenile
- Length at study initiation: 22.9 ± 3.2 mm
- Weight at study initiation: 0.20 ± 0.06 g

ACCLIMATION
- Acclimation period: > 14 days
- Acclimation conditions: same as test
- Type and amount of food during acclimation: Tetramin Flakes and live Brine Shrimp
- Feeding frequency during acclimation: at least once daily; not fed for 48 h prior to testing
- Health during acclimation: No mortalities during 48 hours prior to testing, no treatments for disease
Test type:
semi-static
Water media type:
saltwater
Remarks:
synthetic seawater from artificial sea salts
Limit test:
no
Total exposure duration:
96 h
Test temperature:
22.6 - 22.8 °C (0-23 h)
21.9 - 22.3 °C (24 - 47 h)
22.4 - 22.8 °C (48 - 71 h)
22.3 - 22.8 °C (72 - 96 h)
21.9 - 22.8 °C (complete exposure period)
pH:
8.2 - 8.3 (control)
8.2 - 8.3 (solvent control)
8.1 - 8.3 (4.52 mg a.i./L, mean measured concentration)
8.2 - 8.3 (9.09 mg a.i./L, mean measured concentration)
Dissolved oxygen:
5.9 - 6.7 mg O2/L (control)
5.9 - 6.6 mg O2/L (solvent control)
5.9 - 6.6 mg O2/L (4.52 mg a.i./L, mean measured concentration)
6.0 - 6.7 mg O2/L (9.09 mg a.i./L, mean measured concentration)
Salinity:
22‰ (control)
22‰ (solvent control)
22‰ (4.52 mg a.i./L, mean measured concentration)
22‰ (9.09 mg a.i./L, mean measured concentration)
Nominal and measured concentrations:
control, solvent control, 5.00 and 10.00 mg a.i./L (nominal)
<0.025, <0.025, 4.52 and 9.09 mg a.i./L (mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: aquarium
- Material, size, headspace, fill volume: glass, 38 L (49.5 x 25.4 x 30.5 cm), fill volume: 30 L (49.5 x 25.4 x 23.8 cm)
- Renewal rate of test solution: daily
- No. of organisms per vessel: 30
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.2 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Artificial sea salts (Crystal Sea Marinemix) mixed with process water to produce a salinity of 20 ± 5 parts per thousand (‰). Process water is made by blending reverse osmosis water with dechlorinated municipal water and/or spring water.
- Total organic carbon: 0.25 - 0.79 mg/L
- Intervals of water quality measurement: daily, in old and newly prepared test solutions

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark with 30 min transition period
- Light intensity: 604 to 1021 lux (mean: 867 lux)

EFFECT PARAMETERS MEASURED
- Survival (mortality) and sublethal behavioral effects

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 9.09 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
- Mortality of control: no mortality
- Abnormal responses: no abnormalities
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: precipitants were noted in the 10 mg a.i./L test level
Results with reference substance (positive control):
No reference substance was tested.
Reported statistics and error estimates:
No statistical calculations were conducted to determine the LC50 for this study. The NOEC and LOEC were empirically determined based upon observation data including lethal and sublethal effects.

Measured recoveries ranged from 71 to 119% of nominal.

Mean Measured Recovery Range: 90 to 91% of nominal.

Control Recoveries: Acceptable results and no indication of control contamination. All samples were below the LOQ (0.025 mg a.i./L).

Validity criteria fulfilled:
yes
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Dec 2013 - 17 Mar 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
rev. 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
2008
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Version / remarks:
April 1996
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10 mg a.i./L
- Sampling method: Samples were taken on Day 0 (2013-12-02), Day 2 (2013-12-04) and Day 4 (2013-12-06)
- Sample storage conditions before analysis: Freezer
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test media were prepared by weighing an adequate amount of the test item and dissolving it in DMF and a part of the test water, before pouring it into the test vessels.
- Controls: Control, Solvent Control
- Chemical name of vehicle: Dimethylformamide (DMF)
- Concentration of vehicle in test medium: 0.1 mL/L
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Lot: F19/13
- Source: Hatchery Dr. Rosengarten, Oesede-Georgsmarienhütte; Germany
- Length at study initiation: mean 4.1 cm, SD 0.5 cm
- Weight at study initiation: mean 0.8 g, SD 0.3 g

ACCLIMATION
- Acclimation period: 48 h
- Acclimation conditions: same as test
- Type and amount of food during acclimation: commercial trout food (Inicio, BioMar, Denmark)
- Feeding frequency during acclimation: daily
- Health during acclimation: < 5% mortality

FEEDING DURING TEST
- no feeding
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
40 - 60 mg CaCO3/L
Test temperature:
12.0 - 12.1 °C (control)
12.0 °C (solvent control)
11.8 °C (test concentration)
pH:
6.8 - 7.1 (control)
6.8 - 7.2 (solvent control)
6.7 - 7.3 (test concentration)
Dissolved oxygen:
89 - 100 % (control)
89 - 101 % (solvent control)
86 - 102 % (test concentration)
Conductivity:
<0.2 mS/cm
Nominal and measured concentrations:
control, solvent control, 11.2 mg/L (test item, nominal), corresponding to control, solvent control, 10.0 mg a.i./L (nominal)
<1.25, <1.25, 10.9 mg a.i./L (mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: aquarium
- Type: open
- Material, size, headspace, fill volume: 38 x 32 x 36 cm (h x w x d); test volume: 40 L
- Aeration: Yes, to ensure oxygen saturation
- No. of organisms per vessel: 30
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.60 g fish / L test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was used. Addition of salt stock solution to demineralized water according to ISO guideline.
- Chlorine: <0.01 mg/L
- Intervals of water quality measurement: experimental Day 0 (test initiation), Day 2, Day 4 (test termination)

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark

EFFECT PARAMETERS MEASURED: Cumulative Mortality and Behavioral Observations; at 4 h, 24 h, and every 24 h following

TEST CONCENTRATIONS
- Range finding study: yes, non-GLP
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 11.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: none
- Mortality of control: There was less than 5% mortality within the 48 hour settling-in period and ≤ 10% mortality in the controls.

Analytical results:

The chemical analysis of the test item revealed recoveries between 107 % and 110 % of nominal. The analytical results of the test solution during the 96 hours exposure period demonstrate the stability of the test item concentration over the entire test period. Therefore the results of this study are presented based on the nominal concentrations.

Biological results:

There were neither any sub-lethal effects nor any mortality in the control group and in the single concentration tested.

Validity criteria fulfilled:
yes
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
17 Feb 2014 - 31 Mar 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
Verision: 1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Version / remarks:
Public draft, 1996
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10 mg a.i./L
- Sampling method: Samples were taken on Day 0 (2014-02-17), Day 2 (2014-02-19) and Day 4 (2014-02-21)
- Sample storage conditions before analysis: Freezer
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test media were prepared by weighing an adequate amount of the test item and dissolving it in DMF and a part of the test water, before pouring it into the test vessels.
- Controls: Control, Solvent Control
- Chemical name of vehicle: Dimethylformamide (DMF)
- Concentration of vehicle in test medium: 0.1 mL/L
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnow
- Lot: F20/13A
- Source: brood stock cultured in the test facility at Bayer CropScience AG
- Length at study initiation: mean 3.5 cm, SD 0.3 cm
- Weight at study initiation: mean 0.4 g, SD 0.1 g

ACCLIMATION
- Acclimation period: 48 h
- Acclimation conditions: same as test
- Type of food during acclimation: Inicio (formerly Ecostart 17), BioMar, Denmark
- Feeding frequency during acclimation: daily
- Health during acclimation: < 5% mortality

FEEDING DURING TEST
- no feeding
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
40 - 60 mg CaCO3/L
Test temperature:
22.3 - 22.4 °C (control)
22.3 - 22.5 °C (solvent control)
22.4 - 22.5 °C (test concentration)
pH:
7.4 - 7.5 (control)
7.3 - 7.5 (solvent control)
7.1 - 7.5 (test concentration)
Dissolved oxygen:
83 - 95 % (control)
89 - 94 % (solvent control)
65 - 95 % (test concentration)
Conductivity:
< 0.2 mS/cm
Nominal and measured concentrations:
control, solvent control, 11.2 mg/L (test item, nominal), corresponding to control, solvent control, 10.0 mg a.i./L (nominal)
<0.00625, <0.00500, 10.9 mg a.i./L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: aquarium
- Type: open
- Material, size, headspace, fill volume: 38 x 32 x 36 cm (h x w x d); test volume: 40 L
- Aeration: Yes, to oxygen saturation point
- No. of organisms per vessel: 30
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 0.30 g fish / L test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was used. Addition of salt stock solution to demineralized water according to ISO guideline.
- Chlorine: <0.01 mg/L
- Intervals of water quality measurement: Samples taken on experimental Day 0 (test initiation), Day 2 and Day 4 (test termination)

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark

EFFECT PARAMETERS MEASURED: Cumulative Mortality and Behavioral Observations; at 4 h, 24 h, and every 24 h following

TEST CONCENTRATIONS
- Range finding study: yes, non-GLP
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 11.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality

The analytical determination of the test item (in water by HPLC – MS/MS) revealed recoveries of 107% - 110% of nominal over the whole testing period of 96 h at the limit test concentration of 11.2 mg test item/L (corresponding to 10.0 mg a.s./ L). There were neither any sub-lethal effects nor any mortality in the control group.

Validity criteria fulfilled:
yes

Description of key information

Freshwater:

LC50 (96 h) > 10.0 mg a.s./L (M-486042-01-1, Oncorhynchus mykiss, nominal, OECD 203)

Marine water:

LC50 (96 h) > 9.09 mg a.s./L (M-508088-01-1, Cyprinodon variegatus, measured (not specified), OECD 203)

Key value for chemical safety assessment

LC50 for freshwater fish:
10 mg/L
LC50 for marine water fish:
9.09 mg/L

Additional information

Three studies are available on the short-term toxicity of the test item to fish (M-486042-01-1, M-489576-01-1, M-508088-01-1). The studies were performed according to OECD guideline 203 and GLP, with analytical monitoring of the test item concentration. The mean recovery rates ranged between 90 to 91% and 107% to 110 %, respectively. Different test species were used in the three studies, one with marine (Cyprinodon variegatus) and two with freshwater species (Oncorhynchus mykiss & Pimephales promelas).

The studies with rainbow trout and fathead minnow were conducted as a limit test with the highest test item concentration of 10 mg a.s./L as functional limit of solubility. Due to a moderate water solubility the test item was dissolved in DMF. Thirty fish were exposed in a limit test for 96 h under static test conditions to a nominal concentration of 10.0 mg a.s./L against a water control and a solvent control (DMF, 0.1 mL/L) with further 30 fish each, for both studies. The analytical determination of the test item (in water by HPLC–MS/MS) revealed a mean recovery of 107% - 110% of nominal over the whole testing period of 96 h. The endpoints were reported based on the nominal concentrations. The endpoints for the rainbow trout study and fathead minnow study for limit test showed that 10 mg a.s./L did not cause any mortality neither to rainbow trout (Oncorhynchus mykiss) nor fathead minnow (Pimephales promelas). Therefore, the LC50 (96 h) was > 10.0 mg a.s./ L (practical limit of solubility). None of the 30 fish at this test level showed any symptom after 96 h. The NOEC (96 h) was ≥ 10.0 mg a.s./L based on nominal concentrations.

For the saltwater species sheepshead minnow (30 fish per treatment level) were exposed in a static-renewal system over a period of 96 h to mean measured concentrations of 4.52 and 9.09 mg a.s./L, respectively. The test item was solved in DMF. While precipitants were noted in the 10 mg a.s./L test level, all samples were centrifuged prior to analysis. Measured recoveries ranged from 71 to 119% of nominal concentrations. Mean measured recoveries ranged from 90 to 91% of nominal concentrations. Results are based on mean measured concentrations. The acute effect of the test item on Sheepshead minnow (Cyprinodon variegatus) can be quantified as a LC50 (96h) of > 9.09 mg a.s./L (mean measured concentration), the highest concentration tested.