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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:
08 Aug - 28 Oct 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)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 0.10, 0.22, 0.484, 1.06 and 2.34 mg/L
- Sampling method: Each test concentrations and the control were analytically verified via GC-MS from freshly prepared media after 0, 24, 48 and 72 hours and from corresponding aged test media after 24, 48, 72 and 96 hours. Dissolved organic carbon was determined of the nominal test concentration 2.34 mg/L from freshly prepared media after 0 and from corresponding 24 h aged media after 24 h.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 180 mg/L was freshly prepared with dilution water. Appropriate volumes of the stock solution were pipetted directly into the test vessels filled with appropriate volumes of the dilution water. The test media were mixed with an ultraturrax (1 minute, 12000 rpm). Thereafter, the test vessels were covered with a floating lid. This procedure was repeated for each renewal day.
- Application: Appropriate volumes of the stock solution were pipetted directly into the test vessels.
- Controls: Yes, dilution water without test substance.
- Evidence of undissolved material: No, the test solutions were clear throughout the test period.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Length at study initiation: Average body length at test start: 2.77 cm
- Weight at study initiation: Average body weight at test start: 0.19 g

HOLDING
- Holding conditions: Holding was performed at the test facility at 23 ± 2 °C and diffuse light (7 - 750 Lux, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day.

ACCLIMATION
- Acclimation period: 12 d. The test fish were not be fed 24 h before test start.
- Health during acclimation (any mortality observed): No mortality was observed within the last 7 days before test start.

FEEDING DURING TEST
- Fish were not fed during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
63 mg/L as CaCO3 (control, test start)
Test temperature:
21.6 to 22.1 °C (control)
21.8 to 22.1 °C (test concentration)
pH:
7.24 to 7.79 (control)
7.41 to 7.83 (test concentration)
Dissolved oxygen:
87 to 100 % (control)
75 to 100 % (test concentration)
Conductivity:
175 µS/cm (tap water; measured quarterly)
Nominal and measured concentrations:
Nominal: control, 0.100, 0.220, 0.484, 1.06 and 2.34 mg/L
Geom. mean measured: control, 0.0621, 0.151, 0.278, 0.585 and 1.47 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass aquaria of 13 L, filled with approximately 10 L of dilution water (dimensions: 25.5/18/28 cm, depth of water: 20 cm) and covered with a floating lid (e.g. with aluminum foil).
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): Daily renewal of the test media.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): No vehicle was used.
- Biomass loading rate: Fish density in the test vessels was 0.135 g fish/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine.
- Acidity: 0.20 mmol/L (measured quarterly)
- Acid capacity: 0.80 mmol/L (measured quarterly)
- Culture medium different from test medium: no
- 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. Total hardness of the water was determined at the beginning out of the control. During the test the water temperature was recorded once per hour with a data logger.

OTHER TEST CONDITIONS
- Photoperiod: 10 h photoperiod
- Light intensity: 109 to 140 (mean value 129) Lux

EFFECT PARAMETERS MEASURED: Mortality of fish was recorded. Fish were considered dead if there was no visible movement. Furthermore, visible abnormalities or sublethal effects (e.g. loss of equilibrium, swimming behavior, respiratory function, pigmentation, etc.) were recorded as well.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: yes (at semi-static test conditions)
- Test concentrations: control, 0.180 and 1.80 mg/L
- Results used to determine the conditions for the definitive study: 100% mortality at 1.8 mg/L nominal concentration and 0% mortality at the 0.18 mg/L test item concentration. The test item concentrations of the definitive test were based on the results of this range finding-test.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.205 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
0.278 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
0.151 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality of control: No mortality was observed in the control.
Reported statistics and error estimates:
The LC50-value is given as geometric mean between the concentrations resulting in 0 and 100 % mortality. The concentrations leading to 0 and 100 % mortality (LC0 and LC100) after 96 h were determined directly from the test results. The chosen method for determination of LC50-values were in accordance with the OECD series on testing and assessment No. 54 (2006).

BIOLOGICAL and ANALYTICAL RESULTS:

Table 1: Observations in the Definitive Test (n = 7)

Geometric mean measured test item concentrations [mg/L]

Observation*)

Number of fish effected after different exposure periods [hours]

3

24

48

72

96

1.47

(E)

0

7

7

7

7

 

(2.4)

7

0

0

0

0

0.585

(E)

0

7

7

7

7

 

(1)

7

0

0

0

0

0.278

(E)

0

0

7

7

7

 

(2.2)

0

2

0

0

0

 

(2.1)

0

5

0

0

0

 

(1)

7

0

0

0

0

0.151

(2.5)

0

0

5

5

5

 

(2.4)

0

0

2

2

2

 

(1)

7

7

0

0

0

0.0621

(1)

7

7

7

7

7

Control

(1)

7

7

7

7

7

*) The numbers in brackets correspond to the following observations: (1) = Normal behavior

(2.1) = Lethargy

(2.2) = Side position

(2.4) = Slow escape reflex

(2.5) = Missing escape reflex

(E) = Exitus lethalis

Table 2: Cumulative Mortality [%] in the Definitive Test (n = 7)

Geometric mean measured test item concentrations [mg/L]

Cumulative Mortality [%] after different exposure periods [hours]

 

3

24

48

72

96

1.47

0

100

100

100

100

0.585

0

100

100

100

100

0.278

0

0

100

100

100

0.151

0

0

0

0

0

0.0621

0

0

0

0

0

Control

0

0

0

0

0

Table 3: Measured Concentrations and Percent of the Nominal Concentrations of the Test Item.

Sampling date

2016-10-24 0 hours (Fresh medium)

2016-10-25 24 hours (Old medium)

2016-10-25 24 hours (Fresh medium)

2016-10-26 48 hours (Old medium)

2016-10-26 48 hours (Fresh medium)

Start of analysis

2016-10-24

2016-10-25

2016-10-25

2016-10-26

2016-10-26

CYCLOGALBANAT®

Nominal concentration of the test item [mg/L]

Meas. conc. [mg/L]

%

Meas. conc.[mg/L]

%

Meas.conc. [mg/L]

%

Meas.conc.[mg/L]

%

Meas.conc.[mg/L]

%

2.34

2.10

90

1.03

44

1)

1.06

0.936

88

0.365

34

0.484

0.479

99

0.196

41

0.413

85

0.153

32

1)

0.220

0.195

89

0.129

59

0.191

87

0.128

58

0.179

81

0.100

0.0899

90

0.0556

56

0.0890

89

0.0454

45

0.0853

85

Control

< LOQ

 

< LOQ

 

< LOQ

 

< LOQ

 

< LOQ

 

Meas. conc. = Measured concentration of the test item, enrichment and dilution factor taken into account % = Percent of nominal concentration of the test item

LOQ = Limit of Quantification (0.01 mg/L test item)

1) = Not analyzed due to 100% mortality after 24 and 48 hours, respectively

Table 4: Measured Concentrations and Percent of the Nominal Concentrations of the Test Item.

Sampling date

2016-10-27 72 hours (Old medium)

2016-10-27 72 hours (Fresh medium)

2016-10-28 96 hours (Old medium)

Geometric Mean

Start of analysis

2016-10-27

2016-10-27

2016-10-28

 

 

CYCLOGALBANAT®

Nominal concentration of the test item [mg/L]

Meas. conc.[mg/L]

%

Meas. conc.[mg/L]

%

Meas. conc.[mg/L]

%

[mg/L]

%

2.34

1)

1.47

63

1.06

0.585

55

0.484

0.278

57

0.220

0.107

49

0.186

85

0.125

57

0.151

69

0.100

0.0306

31

0.0825

82

0.0508

51

0.0621

62

Control

< LOQ

< LOQ

< LOQ

< LOQ

Meas. cone. = Measured concentration of the test item, enrichment and dilution factor taken into account % = Percent of nominal concentration of the test item

LOQ = Limit of Quantification (0.01 mg/L test item)

1) = Not analyzed due to 100% mortality after 24 and 48 hours, respectively

Table 5: Measured Organic Carbon Concentrations of the Test Item Concentration 2.34 mg/L after 0 and 24 hours (measured as DOC, mean value of two measurements).

Sampling date

2016-10-24 0 h (Fresh medium)

2016-10-25 24 h (Old medium)

Nominal test item concentration [mg/L]

CYCLOGALBANAT®

Measured concentration [mg C/L]

Measured concentration [mg C/L]

2.34

5.62

4.91

VALIDITY CRITERIA:

Table 1: Validity criteria for OECD 203.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test

No mortalities in the control

yes

The dissolved oxygen concentration must have been at least 60 per cent of the air

saturation value throughout the test

>75%

yes

There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the

nominal concentration throughout the test. If the deviation from the nominal

concentration is greater than 20 per cent, results should be based on the measured

concentration.

Results are based on measured concentrations

yes

The pH value in the control group did not deviate by more than one unit over the test period.

pH was between 7.24 and 7.83

yes

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
In this study the substance was found to cause lethal effects to zebrafish after 96 h at a geometric mean measured test item concentration of 0.278 mg/L. The LC50 after 96 h was 0.205 mg test item/L (geometric mean of LC0 and LC100). The LC0 and LC100 after 96 h was 0.151 and 0.278 mg test item/L (geometric mean measured test item concentrations).
Executive summary:

The short-term toxicity of the substance towards fish was conducted according to GLP and OECD guideline 203. In a semi-static system with daily renewal of the test media, Danio rerio as the test organism was exposed to the following nominal five test item concentrations: 0.100, 0.220, 0.484, 1.06 and 2.34 mg/L (nominal) corresponding to geometric mean measured concentrations of 0.0621, 0.151, 0.278, 0.585 and 1.47 mg/L. The duration of the test was 96 h. The measured concentrations in freshly prepared media were in the range 81 and 99% of the nominal values. In corresponding 24 h aged test media, the measured concentrations were between 31 and 59% of the nominal values. Therefore, all effect values are based on the geometric mean measured concentrations of the test item. During the 96-h exposure period, no death and visible toxic signs were observed in the control group and 0.0621 mg/L test group, the fish exposed to other 4 test groups showed visible toxic signs such as lethargy, side position, slow or no escape reflex and deaths. The LC50 (96 h) of 0.205 mg/L (geom. mean meas.) was derived from the LC0 (96 h) = 0.151 mg/L and LC100 (96 h) = 0.278 mg/L.

Description of key information

LC50 (96 h) = 0.205 mg/L (geometric mean, Danio rerio, OECD 203)

Key value for chemical safety assessment

LC50 for freshwater fish:
0.205 mg/L

Additional information

One study is available, in which the short-term toxicity of the substance towards fish was investigated. The study was conducted according to GLP and OECD guideline 203. In a semi-static system with daily renewal of the test media, Danio rerio as the test organism was exposed to the following nominal five test item concentrations: 0.100, 0.220, 0.484, 1.06 and 2.34 mg/L (nominal) corresponding to geometric mean measured concentrations of 0.0621, 0.151, 0.278, 0.585 and 1.47 mg/L. The duration of the test was 96 h. The measured concentrations in freshly prepared media were in the range 81 and 99% of the nominal values. In corresponding 24 h aged test media, the measured concentrations were between 31 and 59% of the nominal values. Therefore, all effect values are based on the geometric mean measured concentrations of the test item. During the 96-h exposure period, no death and visible toxic signs were observed in the control group and 0.0621 mg/L test group, the fish exposed to other 4 test groups showed visible toxic signs such as lethargy, side position, slow or no escape reflex and deaths. The LC50 (96 h) of 0.205 mg/L (geom. mean meas.) was derived from the LC0 (96 h) = 0.151 mg/L and LC100 (96 h) = 0.278 mg/L.