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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Jun - 11 Jun 2016
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
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 1.06, 2.13, 4.25, 8.50 and 17 mg/L
- Sampling method: Samples were analysed from freshly prepared media after 0, 24, 48 and 72 hours and from corresponding aged test media after 24, 48, 72 and 96 hours.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Appropriate volumes of the test item were pipetted directly into the test vessels filled with appropriate volumes of the dilution water. Test media were mixed with an ultraturrax (2 min at 8500 rpm). The test vessels were covered with a floating lid. After a settling period of 2 h the fish were inserted. This procedure was repeated for each renewal day.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium conrol
- Evidence of undissolved material: no, test solutions were clear
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Length at study initiation: 2.53 cm (mean)
- Weight at study initiation: 0.141 g (mean)

ACCLIMATION
- Acclimation period: at least 12 d
- Acclimation conditions: same as test
- Type and amount of food during acclimation: Sera Vipan, Sera GmbH, Heinsberg, Germany
- Feeding frequency during acclimation: 3 time a week (4% of the fish body weight per feeding day); not fed 24 h before study initiation
- Health during acclimation: No mortality was observed during this time. No disease treatments were administered throughout holding and testing.

FEEDING DURING TEST
- None
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
59 mg/L as CaCO3
Test temperature:
22.9 - 23.9 °C (mean: 23.3 °C ± 0.3 °C)
pH:
6.97 - 7.57
Dissolved oxygen:
86 - 100% O2 saturation
Nominal and measured concentrations:
nominal: control, 1.06, 2.13, 4.25, 8.50 and 17 mg/L
geometric mean measured: < LOQ, 0.301, 0.462, 1.26, 3.10, 9.30 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: aquaria
- Type (delete if not applicable): covered with a floating lid
- Material, size, headspace, fill volume: Glass, 13 L (25.5/18/28 cm), headspace: 3 L, fill volume: 10 L
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): every 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.099 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water of local origin
- Hardness: 10 - 250 mg CaCO3/L
- Conductivity: 168 µS/cm
- Acidity: 0.10 mmol/L
- Acid capacity: 0.60 mmol/L
- Culture medium different from test medium: same as test
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all test vessels at the beginning and at the end of the exposure and every 24 h from fresh and old test media. Water temperature was recorded once per hour with a data logger. Light intensity was measured on the water surface at test start.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: 7 - 750 lux

EFFECT PARAMETERS MEASURED
- Mortality was recorded after 2, 24, 48, 72 and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: control, 17 mg/L
- Results used to determine the conditions for the definitive study: 100% mortality was recorded after 24 h at the limit concentration.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
6.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI
Remarks:
3.10 - 9.30 mg/L
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
3.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 9.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: Up to a concentration of 3.1 mg/L all fish behaved normal. At the highest test concentration surviving fish were observed to be lethargic, staggering, on a side position and/or missing escape reflex.
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, test solutions were clear
Reported statistics and error estimates:
LC50 value was calculated by linear regression.

Table 1: Cumulative mortality

Geometric mean measured test item concentrations [mg/L]

Cumulative mortality [%] during the study [h]

2

24

48

72

96

9.30

0

0

29

71

71

3.10

0

0

0

0

0

1.26

0

0

0

0

0

0.452

0

0

0

0

0

0.301

0

0

0

0

0

Control

0

0

0

0

0

Table 2: Measured concentrations of the test substance

Nominal concentration

0 h (fresh)

24 h (old)

24 (fresh)

48 h (old)

48 h (fresh)

72 h (old)

72 h (fresh)

96 h (old)

Geometric mean [mg/L]

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

17

11.8

69

9.10

54

14.6

86

11.2

66

9.85

58

9.26

54

5.05

30

6.91

41

9.30

8.5

4.97

58

3.23

38

2.58

30

2.13

25

3.68

43

2.55

30

3.29

39

3.15

37

3.10

4.25

2.56

60

1.23

29

1.53

36

1.09

26

1.17

28

0.810

19

1.24

29

1.01

24

1.26

2.13

0.833

39

0.345

16

0.827

39

0.480

23

0.291

14

0.187

9

0.584

27

0.477

22

0.452

1.06

0.587

55

0.231

22

0.371

35

0.157

15

0.498

47

0.296

28

0.256

24

0.223

21

0.301

Control

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

Table 3: Measured concentrations of the metabolite

Nominal concentration

0 h (fresh)

24 h (old)

24 (fresh)

48 h (old)

48 h (fresh)

72 h (old)

72 h (fresh)

96 h (old)

Geometric mean [mg/L]

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

Meas. [mg/L]

%

17

< LOQ

<1

0.790

6

0.0397

<1

0.599

5

0.0425

<1

0.619

5

0.0383

<1

0.142

1

0.203

8.5

0.0955

1

1.06

16

0.00953

<1

0.349

5

0.0434

<1

0.684

10

0.0192

<1

0.285

4

0.157

4.25

0.00393

<1

0.236

7

0.0166

<1

0.157

5

0.0122

<1

0.214

6

0.0128

<1

0.170

5

0.0787

2.13

< LOQ

<1

0.150

9

0.0222

1

0.199

12

0.00546

<1

0.102

6

0.0101

<1

0.130

8

0.0460

1.06

0.00836

1

0.0786

9

0.0214

3

0.1253

15

0.00791

1

0.0998

12

0.00999

1

0.0792

10

0.0406

Control

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

Validity criteria fulfilled:
yes
Conclusions:
This short-term study with fish resulted in a LC50 (96 h) of 6.72 mg/L (geom. mean measured concentration).

Description of key information

LC50 (96 h) = 6.72 mg/L (measured, OECD 203) 

Key value for chemical safety assessment

LC50 for freshwater fish:
6.72 mg/L

Additional information

The key study with the test item was conducted according to OECD 203 (GLP). Danio rerio was exposed to nominal concentrations of 1.06, 2.13, 4.25, 8.50 and 17 mg/L for 96 h under semi-static conditions. The exposure concentrations were verified by GC/MS analysis. Measurements indicate that the measured concentrations did not remain within 80-120% of nominal. The recovery rate ranged from 14 - 86% in the freshly prepared media and 9 - 66% in the aged test solutions. Thus, the effect concentrations were based on the geometric mean of measured concentrations. After 96 h an LC50 of 6.72 mg/L was calculated by regression analysis. 

Since no marine toxicity tests are available, the data generated with freshwater species were also used for the hazard assessment for the marine aquatic environment.