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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:
2016-06-06 - 2016-07-11 (experimental phase)
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:
as of 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D65189 Wiesbaden
Specific details on test material used for the study:
Stability under test conditions: A supporting chemical analysis was performed within this study.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 20, 40, 80, 100 mg/L
- Sample storage conditions before analysis: The supporting analyses could not be performed on the same day of collection of the specimens. Therefore, these specimens from freshly prepared test solutions were stored deep-frozen, thawed over-night and then analysed.
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was tested at concentrations 10 to 100 mg/L (main test) prepared in drinking water from the municipal drink water supply entering the institute. The drinking water used for the test refers to the OECD Guideline 203 and is controlled regularly (monthly). The test item was introduced into the water by direct weighing into the different treatments to give the final nominal concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not stated
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Source: Aquahandel.de (Holtenser Weg 6a, 37186 Moringen)
- Length at study initiation (length definition, mean, range and SD): length ≈ 2.0 ±1.0 cm

ACCLIMATION
- Feeding frequency during acclimation: The fish were not fed 24 hr prior to testing.

FEEDING DURING TEST
The fish were not fed 24 hr prior to testing and during the whole test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
none
Hardness:
2.01 / 0.72 mmol/L (Ca2+ / Mg2+) (average Jan-Dec 2015)
1.84 / 0.69mmol/L (Ca2+ / Mg2+) (average Jan-Jul 2016)
Test temperature:
21.0 to 22.6 °C
pH:
pH of the test solutions was not adjusted.
pH = 7.81 - 8.64
Dissolved oxygen:
8.0 - 8.4 mg/L
Nominal and measured concentrations:
Nominal: 0, 10, 20, 40, 80, 100 mg/L
Measured (after correction): 8.7-11.7, 20.1-21.9, 38.6-42.7, 71.1-77.4, 89.4-109.9 mg/L
Details on test conditions:
TEST SYSTEM
- Aeration: The test solutions were aerated during the incubation period (96 hr) in order to verify an oxygen content of >60% of the saturation value during the study
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): daily

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Intervals of water quality measurement: monthly

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : behaviour, mortality (daily)

TEST CONCENTRATIONS
- Test concentrations: 120 mg/L
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
<= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: At 80 mg/L and 100 mg/L animals disimpassioned and partially at the bottom of the aquarium and increased movement of gills, lesser agile and swimming at the bottom
- Mortality of control: none
- Other adverse effects control: none stated
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
n/a

Results – Biological Part

Screening-Test (Concentrations successively tested)

 

Nominal Concentration

[mg Test Item per Liter of Tap Water]

Mortality [%] after

1d (3 hr)

2d

3d

4d

1201

33 (1 of 3)

67 (2 of 3)

67 (2 of 3)

67 (2 of 3)

1) Erroneously the technician used a wrong measuring cylinder resulting in a smaller final volume (2.5L instead of 3L) and thus in a higher concentration than intended in the study plan (120 instead of 100 mg/L). This represents a deviation of the study plan which is not considered to have an influence on the integrity of the study.

 

Accompanying Determinations (Screening-Test, semistatic Test Procedure):

Nominal conc. of Test Item [mg/L]

Incubation time[h]

pH-Value

O2-Content [mg O2/L]

Temperature [°C]

t01)

(t24hr)2)

t01)

(t24hr)2)

t01)

(t24hr)2)

120

0

24

48

72

96

8.12

8.12

8.10

8.11

-

-

8.53

8.55

8.68

8.61

8.5

8.4

8.4

8.3

-

-

8.3

8.4

8.5

8.3

21.8

21.2

21.1

21.4

-

-

21.2

21.1

21.2

21.1

 

1)Values in the fresh test solutions, respectively;2)after 24 hr of incubation with the fish

 

LC0(after 96 hr)    <  120 mg/L of tap-water

LC50(after 96 hr)      <  120 mg/L of tap-water

LC100(after 96 hr)      >  120 mg/L of tap-water

 

Nominal Concentration

[mg Test Item per Liter of Tap Water]

Mortality [%] after

0 d

2d

3d

4d

12.5 (10)1

0 (0 of 3)

0 (0 of 3)

0 (0 of 3)

0 (0 of 3)

 

1) Erroneously, within the first three days the technician used a wrong measuring cylinder resulting in a smaller final volume (2.5L instead of 3L) and thus in a higher concentration than intended in the study plan (12.5 instead of 10 mg/L). This represents a deviation of the study plan which is not considered to have an influence on the integrity of the study.

 

Resulting:

LC0(after 96h)      >  10 mg/L of tap-water

LC50(after 96h)         >  10 mg/L of tap-water

LC100(after 96h)         >  10 mg/L of tap-water

 

Accompanying Determinations:

Nominal conc. of Test Item [mg/L]

Incubation time [hr]

pH-Value

O2-Content [mg O2/L]

Temperature [°C]

t01)

(t24hr)2)

t01)

(t24hr)2)

t01)

(t24hr)2)

 

12.5 (10)1

0

25

50.5

72

96

8.13

8.15

8.13

8.18

-

-

8.53

8.64

8.60

8.63

8.3

8.3

8.4

8.3

-

-

8.7

8.6

8.5

8.4

21.3

21.2

21.5

21.6

-

-

21.1

21.2

21.1

21.0

 

1)Values in the fresh test solutions, respectively;2)after 24 hr of incubation with the fish

 

Results-Main Test

 

Nominal Concentration

[mg Test Item per Liter of Tap Water]

Mortality [%] after

1d

2d

3d

4d (96 hr)

Control (0)

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

10

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

0 (0of 7)

20

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

40

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

80

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

100

0 (0 of 7)

0 (0 of 7)

0 (0 of 7)

14.3 (1 of 7)

 

 

Accompanying determinations (Main Test, semistatic Test Procedure):

 

 

Nominal conc. of Test Item [mg/L]

Incubation time[d]

pH-Value

O2-Content [mg O2/L]

Temperature [°C]

t01)

(t24hr)2)

t01)

(t24hr)2)

t01)

(t24hr)2)

 

 

Control (0)

0

1

2

3

4 (96hr)

8.09

7.81

8.03

8.13

-

-

8.56

8.64

8.63

8.58

8.2

8.4

8.2

8.4

-

-

8.4

8.3

8.3

8.3

21.8

21.3

21.5

21.4

-

-

22.6

21.9

22.5

22.3

10

0

1

2

3

4 (96hr)

8.10

7.81

8.09

8.09

-

-

8.57

8.35

8.61

8.54

8.2

8.3

8.3

8.4

-

-

8.4

8.3

8.2

8.3

21.7

21.2

21.5

21.0

-

-

22.5

21.9

22.4

22.3

20

0

1

2

3

4 (96hr)

8.11

0.03

8.10

8.09

-

-

8.58

8.63

8.61

8.60

8.2

8.2

8.3

8.3

-

-

8.2

8.3

8.2

8.3

22.0

21.3

21.1

21.4

-

-

22.5

21.9

22.5

22.3

40

0

1

2

3

4 (96hr)

8.12

8.05

8.10

8.09

-

-

8.57

8.49

8.55

8.54

8.2

8.3

8.3

8.4

-

-

8.2

8.3

8.3

8.3

22.1

21.7

21.1

21.7

-

-

22.6

21.9

22.6

22.3

80

0

1

2

3

4 (96hr)

8.12

8.03

8.09

8.09

-

-

8.56

8.55

8.48

8.50

8.0

8.2

8.3

8.3

-

8.3

8.3

8.2

8.2

22.2

21.2

21.3

21.1

-

-

22.5

22.0

22.5

22.3

100

0

1

2

3

4 (96hr)

8.13

8.05

8.11

8.09

-

-

8.64

8.65

8.47

8.50

8.1

8.3

8.3

8.2

-

8.3

8.3

8.3

8.2

22.2

21.1

21.3

21.2

-

-

22.5

21.9

22.5

22.4

1)Values in the fresh test solutions, respectively;2)after 24hr of incubation with the fish

 

 

Assumed Toxicity Values (after 96h):

 

On the Basis of the nominal Concentrations [mg/L]

                                          LC10        156.7

           95%-CL                 lower          n.d.

                                         upper          n.d.

                       

                                          LC20        >100

           95%-CL                 lower          n.d.

                                         upper          n.d.

                                                                  

                                          LC50        >100

           95%-CL                 lower          n.d.

                                         upper          n.d.

                                                                  

          Mortality                LOEC       = 100

                                        NOEC        100

 

 

Observations on the behaviour of the fish (Main Test):

 

Time (h)

Effects / Observations

1

At 80 mg/L and 100 mg/L animals disimpassioned and partially at the bottom of the aquarium and increased movement of gills

2

At 80 mg/L and 100 mg/L animals disimpassioned and partially at the bottom of the aquarium and increased movement of gills; animals at other test concentrations without symptoms

4 – 6

At 80 mg/L and 100 mg/L animals disimpassioned and partially at the bottom of the aquarium and increased movement of gills; animals at other test concentrations without symptoms

18 – 20

At 80 mg/L and 100 mg/L animals disimpassioned and partially at the bottom of the aquarium and increased movement of gills; animals at other test concentrations without symptoms

24

At 80 mg/L and 100 mg/L animals disimpassioned and partially at the bottom of the aquarium and increased movement of gills; animals at other test concentrations without symptoms

28 – 30

At 80 mg/L and 100 mg/L animals lesser agile and swimming at the bottom

42 – 44

At 80 mg/L and 100 mg/L animals lesser agile and swimming at the bottom, at 100 mg/L one fish without movements in one corner of the aquarium

48

At 80 mg/L and 100 mg/L animals lesser agile and swimming at the bottom, at 100 mg/L one fish with red discoloured head

48-66

At 80 mg/L and 100 mg/L animals lesser agile and swimming at the bottom, at 100 mg/L one fish with red discoloured head

72

At 80 mg/L and 100 mg/L animals lesser agile and swimming at the bottom, at 100 mg/L 2 fish apathetically and one of them disoriented

96

At 100 mg/L one fish dead and one without movements in one corner of the aquarium

 

 

Results of the Analytical Part (GC-MS)

The results of the supporting analyses can be summarised as follows:

Sample Designation

mg test item/L found

mg test item/L found after “procedural recovery” correction

% Recovery on basis of Nominal Concentrations

% Recovery on Basis of t0

Control time t0

n.d.

-

-

-

10 mg/L time t0

6.95

8.7 (a)

87.4

-

20 mg/L time t0

15.96

20.1 (a)

100.4

-

40 mg/L time t0

32.75

42.7 (b)

106.7

-

80 mg/L time t0

54.95

71.6 (b)

89.6

-

100 mg/L time t0

84.28

109.9 (b)

109.9

-

Control time t24 hr

n.d.

-

-

-

10 mg/L time t24 hr

6.97

8.8 (a)

87.7

100.3

20 mg/L time t24 hr

17.42

21.9 (a)

109.6

109.1

40 mg/L time t24 hr

29.63

38.6 (b)

96.6

90.5

80 mg/L time t24hr

54.50

71.1 (b)

88.8

99.2

100 mg/L time t24 hr

72.06

94.0 (b)

94.0

85.5

Control time t0

n.d.

-

-

-

10 mg/L time t0

8.58

11.7 (c)

117.2

-

20 mg/L time t0

15.19

20.8 (c)

103.8

-

40 mg/L time t0

34.01

41.7 (d)

104.3

-

80 mg/L time t0

63.09

77.4 (d)

96.7

-

100 mg/L time t0

76.46

93.8 (d)

93.8

-

Control time t24 hr

n.d.

-

-

-

10 mg/L time t24 hr

6.79

9.3 (c)

92.8

79.1

20 mg/L time t24 hr

15.43

21.1 (c)

105.4

101.6

40 mg/L time t24 hr

33.81

41.5 (d)

103.7

99.4

80 mg/L time t24 hr

60.76

74.5 (d)

93.2

96.3

100 mg/L time t24 hr

72.89

89.4 (d)

89.4

115.5

Spike 10 mg/L
("Procedural Recovery"), 1stSer.

7.95 (a)

(10)

-

-

Spike 100 mg/L
("Procedural Recovery"), 1stSer.

76.70 (b)

(100)

-

-

Spike 10 mg/L
("Procedural Recovery"), 2ndSer.

7.32 (c)

(10)

-

-

Spike 100 mg/L
("Procedural Recovery"), 2ndSer.

81.53 (d)

(100)

-

-

 

(a), (b),(c), (d) are the respective bases for the calculations with respect of the “procedural recoveries”.

As a result of the supporting analyse it can be stated that the results of the biological part should be based on the nominal concentrations applied because the necessary stability of ≥ 80% could be shown. The low % recovery on basis of the t0value at concentration 10 mg/L of the second series is due to the high recovery at t0. The geometric mean of t0and after 24 hr of that pair of values is >100% of the nominal concentration and thus, it can be stated that the fish were always in contact with >80% of the nominal concentrations applied, and therefore the results of the biological part shall be based on the nominal concentrations applied.

 

Validity of Results

The criteria of validity of results given by the OECD Guideline No. 203 (1992) were kept within this study. In the control no dead animals were observed. In the test solutions the oxygen concen­tration did not drop below a value of 60% of the saturation value (»5 mg O2/L).

 

 

Discussion

The test item was tested for acute toxicity towards fish according to OECD Guideline No. 203 (1992) using zebra fish. In order to investigate the influence of the test item towards the fish, the animals were exposed to up to 120 mg (screening) of the test item per litre of test solution. Under the conditions used for the test, no significant toxic effects of the test item towards fish were observed at 100 mg/L (main test). As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation. Based on this, the reported effect concentrations should refer to the nominal concentrations tested. The following LC-values (effective concentrations) were determined accordingly:

 

On the Basis of the nominal Concentrations [mg/L]

                                          LC10        156.7

           95%-CL                 lower          n.d.

                                         upper          n.d.

                       

                                          LC20        >100

           95%-CL                 lower          n.d.

                                         upper          n.d.

                                                                  

                                          LC50        >100

           95%-CL                 lower          n.d.

                                         upper          n.d.

                                                                  

          Mortality                LOEC       = 100

                                        NOEC        100

 

The criteria of validity of results given by the OECD Guideline 203 were kept within this study. In the control no dead animals were observed. In the test solutions the oxygen concentration did not drop below a value of 60% of the saturation value (»5 mg O2/L).

 

Validity criteria fulfilled:
yes
Conclusions:
The study was performed according to OECD TG 203 with no relevant deviations, the results were so obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable: Under the conditions used for the test, no significant toxic effects of the test item towards fish were observed at 100 mg/L (main test). As a conclusion of the analytical part of this study, it can be stated that the concentrations of the test item remained sufficiently stable during incubation. Based on this, the reported effect concentrations should refer to the nominal concentrations tested. The following LC-values (effective concentrations) were determined accordingly:
Mortality
LOEC = 100 mg/L
NOEC ≤ 100 mg/L
LC50 > 100 mg/L
Based on these results, the test item does not need to be classified as acute or chronic toxic to the aquatic environment.
Executive summary:

3-Methyl-thiazolidin-thion-2 was tested for acute toxicity towards fish according to OECD Guideline No. 203 (1992) using zebra fish. In order to in­vestigate the influence of the test item towards the fish, the animals were exposed to up to 120 mg (screening) of the test item per litre of test solution. Under the conditions used for the test, no significant toxic effects of the test item towards fish were observed at 100 mg/L (main test). As a conclusion of the analytical part of this study, it can be stated that the concentrations of 3-Methyl-thiazolidin-thion-2 remained sufficiently stable during incubation. Based on this, the reported effect concentrations should refer to the nominal concentrations tested. The following LC-values (effective concentrations) were determined accordingly:

On the Basis of the nominal Concentrations [mg/L]

                                          LC10        156.7

           95%-CL                 lower          n.d.

                                         upper          n.d.

                       

                                          LC20        >100

           95%-CL                 lower          n.d.

                                         upper          n.d.

                                                                  

                                          LC50        >100

           95%-CL                 lower          n.d.

                                         upper          n.d.

                                                                  

          Mortality                LOEC       = 100

                                        NOEC        100

 

The criteria of validity of results given by the OECD Guideline 203 were kept within this study. In the control no dead animals were observed. In the test solutions the oxygen concen­tration did not drop below a value of 60% of the saturation value (»5 mg O2/L).

Description of key information

Acute toxicity to fish: LC50(96h) > 100 mg/L for Brachidanio rerio (OECD 203, GLP, semi-static)

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

The result LC50(96h) > 100 mg/L does not trigger classification as harmful for the aquatic environment, neither acute nor chronic, according to Regulation (EC) 1272/2008.