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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
None.
Analytical monitoring:
yes
Details on sampling:
The content of the test item in one replicate per concentration and the control was measured at the beginning and at the end and during the running test once a week changing systematically amongst replicates, before and after addition of the stock solution, but all replicates were analysed at least once. Samples were analysed for test item concentration immediately after sampling.
Vehicle:
no
Details on test solutions:
Before use in the test, the test item was melted at 32 ± 3 °C in a water bath. Stock solutions containing 35 mg/L in dilution water were freshly prepared daily. For this, 35 µL/L of the melted test item was added on the surface of tempered (32 ± 3 °C) dilution water and stirred on a magnetic stirrer until complete dissolution. The concentrations to be tested were prepared by addition of the necessary amount of stock solution into the aquaria.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Fertilised eggs of Danio rerio HAMILTON-BUCHANAN (common name Zebrafish) were used in the test.

Parents and Husbandry
Parents obtained from Umweltbundesamt (UBA, Germany environmental authority) and used for breeding and production of eggs.
Vessels glass aquaria , 30 x 30 x 60 cm, water volume approx..50 L
Medium chlorine-free tap water with a copper concentration below 0.05 mg/L, aerated with glass tubes
Food three times a working day with warm-water fish food (powdered flake food, trade name sera vipan flake food, one spoon per feeding) and living Daphnia magna (not quantified)
Cleaning at least once a week
Medium renewal monthly
Photoperiod 12/12 hours
Light intensity 500 lux (Osram L 18 W/840 tubes, day white spectrum)
Temperature 23 (21 - 25°C min - max)

Production of Eggs
For production of eggs, the spawn dishes were placed into the tempered aquaria where temperature was raised to 28 ± 1°C one day before the start of the test. Five aquaria were used. In each aquaria were placed two males and one female fish. On the next morning, after spawning of the adult animals, about 45 minutes after lighting, the eggs were removed from the spawning aquaria with a pipette and placed in dishes filled with dilution water. Then the eggs were checked via microscope for fertilization. Viable fertilized eggs were separated from unfertilized eggs and 20 fertilized eggs per replicate were transferred in a small stainless steel dish, filled with dilution water .

Food
See table 1 of M&M.

Production of Artemia
2 L of a stock solution containing 20 g/L NaCL were filled in a glass flask. Then 4 dosage spoons Artemia nauplii eggs (JBL Artemio ® Pur) were added. This solution was aerated at room temperature for 48 ± 1 hours. Then the solution was transferred in a separating funnel. About 2 cm above the outlet, the separating funnel was coated with black foil. About 15 minutes later, the artemia are cumulated in the light zone below the foil. Then, the artemia were removed carefully. Before usage, the artemia were washed several times with tap water .
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Remarks on exposure duration:
None.
Post exposure observation period:
None.
Hardness:
106.8 mg CaCO3/L at beginning and end of the study.
Test temperature:
25.0°C - 26.9°C (min - max)
pH:
7.7 - 8.2 (min - max)
Dissolved oxygen:
6.0 - 8.3 mg/L (min - max)
Salinity:
Freshwater
Conductivity:
Not measured.
Nominal and measured concentrations:
Nominal: 0 ; 0.22 ; 0.46 ; 1 ; 2.2 ; 4.6 mg/L
Measured (arithmetic means of geometric means): 0 ; 0.19 ; 0.26 ; 0.41 ; 0.66 ; 0.88 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: stainless steel aquaria with outlet
- Material, size, headspace, fill volume: 15 L capacity (12-L medium)
- Aeration: yes, from day 14 until the end of the study
- Method: semi-static (one third renewal every day, including week-ends)
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: drinking water
- Total organic carbon: 0.5 mg/L
- Metals: < 2 µg/L
- Pesticides: < LOD
- Chlorinated organic compounds: < LOD
- pH: 8.05
- Ca/mg ratio: 17/2
- Salinity: < 10 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12L:12D
- Light intensity: 500 lux
- Spectrum: day light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Hatching
- Larval survival
- Larval mean weight per replicate
- Larval length
Reference substance (positive control):
not required
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.66 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
number hatched
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.66 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
weight
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
length
Details on results:
Results on hatching and survival rates and final weight and length of larvae are presented in tables 1 to 4.
Results with reference substance (positive control):
None.
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Excel®). Hatching and survival rates, as well as final larval weight and length were analysed with the ToxRat® Professional software, version 3.2.1.

Table 1: Hatching Data

Nominal

Concentration

 inmg/L

Replicate

Number of Fish at the Start

Number of Fish

hatched

Hatching

rate in %

Mean 

in %

Blank control

1

20

18

90

88

2

20

17

85

3

20

17

85

4

20

18

90

0.22

1

20

18

90

84

2

20

17

85

3

20

16

80

4

20

16

80

0.46

1

20

17

85

89

2

20

19

95

3

20

19

95

4

20

16

80

1

1

20

17

85

85

2

20

15

75

3

20

20

100

4

20

16

80

2.2

1

20

17

85

83

2

20

15

75

3

20

18

90

4

20

16

80

4.6

1

20

8

40

40

2

20

9

45

3

20

8

40

4

20

7

35

 

Table 2: Post–hatch survival

Nominal

Concentration

 inmg/L

Replicate

Number of Fish

hatched

Number of Fish at the End

Post - hatch survival

in %

Mean

in %

Blank control

1

18

17

94

86

2

17

15

88

3

17

14

82

4

18

14

78

0.22

1

18

16

89

88

2

17

13

76

3

16

14

88

4

16

16

100

0.46

1

17

15

88

85

2

19

16

84

3

19

13

68

4

16

16

100

1

1

17

17

100

87

2

15

12

80

3

20

17

85

4

16

13

81

2.2

1

17

15

88

83

2

15

12

80

3

18

14

78

4

16

14

88

4.6

1

8

4

50

48

2

9

3

33

3

8

4

50

4

7

4

57

 

Table 3: Weight of Surviving Fish

Nominal

Concentration

 inmg/L

Replicate

Mean Weight per Replicate

in mg

Mean Weight compared to Control
in %

Blank control

1

9.8

--

2

10.8

3

11.6

4

11.9

0.22

1

9.3

94

2

11.1

3

10.8

4

10.2

0.46

1

12.3

110

2

10.9

3

12.3

4

12.9

1

1

10.6

110

2

12.5

3

11,9

4

13.3

2.2

1

12.4

104

2

11.8

3

11.2

4

10.3

4.6

1

11.7

143

2

17.5

3

17.9

4

16.0

 

Table 4: Length of Surviving Fish

Nominal

Concentration

 inmg/L

Replicate

Mean Length per Replicate

in mm

Length compared to Control
in %

Blank control

1

9.9

--

2

10.4

3

10.6

4

10.3

0.22

1

9.9

98

2

10.5

3

9.2

4

10.1

0.46

1

10.2

93

2

9.3

3

8.8

4

10.1

1

1

9.7

105

2

11.0

3

11.3

4

11.3

2.2

1

10.5

102

2

10.4

3

9.9

4

10.4

4.6

1

10.0

111

2

11.3

3

12.3

4

12.3

 



Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test item in the Early-Life Stage Toxicity Test against Danio rerio was tested. The study was performed using 5 treatments ranging from 0.22 mg/L to 4.6 mg/L nominal concentration. Adverse effects were observed only in the highest concentrated treatment. Hatching rate in the highest treatment (4.6 mg/L nominal ; 0.88 mg/L estimated) was 40% while it was 88% in the control group. Larval survival rate (calculated on the number of hatched larvae) was 48% while it was 86% in the control group. Larval growth (length and weight) remained unaffected in all treatments. NOEC was 2.2 mg/L (nominal), i.e. 0.66 mg/L estimated.
Executive summary:

The toxicity of the test item in the Early-Life Stage Toxicity Test against Danio rerio was tested. The study was performed using 5 treatments ranging from 0.22 mg/L to 4.6 mg/L nominal concentration. All control and treatment groups were tested in quadruplicate with 20 eggs in each replicate.

Because the test item is poorly water soluble and not stable in aqueous medium, adjustment of stable test concentrations under flow-through conditions was not possible. Therefore, the study was performed under semi-static conditions in 15-L stainless vessels filled with 12 L test medium. Every day (including week-ends), freshly prepared stock solutions were added into the aquaria to adjust the target concentrations. Additionally, to keep good water quality during the study, before addition of the stock solution, every day at least 4 L tempered fresh dilution water was added slowly opposite to the outlet in each aquaria. Hatching and larval survival were recorded everyday throughout the study. At termination of the study (D35), the larvae were weighted blotted dry on a replicate basis. In addition, length was measured in all live larvae at D35.

The concentrations of the test item in the blank control and in one replicate per treatment was measured once a week, before and after addition of the stock solution. Daily exposure was estimated from the geometric mean of the measured concentrations before addition of both fresh dilution water and stock solution and after addition of the stock solution. The biological results were based on the arithmetic mean of the six geometric concentrations estimated for each of the six sampling days. Because no test item could be detected before addition of the stock solution, the limit of detection of 0.2 mg/L was used for calculation of the geometric means, in agreement with the OECD guidance document no. 23 on aquatic toxicity testing of difficult substances and mixtures.

Adverse effects were observed only in the highest concentrated treatment. Hatching rate in the highest treatment (4.6 mg/L nominal ; 0.88 mg/L estimated) was 40% while it was 88% in the control group. Larval survival rate (calculated on the number of hatched larvae) was 48% while it was 86% in the control group. Larval growth (length and weight) remained unaffected in all treatments. NOEC was 2.2 mg/L (nominal), i.e. 0.66 mg/L estimated.

Description of key information

The toxicity of the test item in the Early-Life Stage Toxicity Test against Danio rerio was tested. The study was performed using 5 treatments ranging from 0.22 mg/L to 4.6 mg/L nominal concentration. All control and treatment groups were tested in quadruplicate with 20 eggs in each replicate.

Because the test item is poorly water soluble and not stable in aqueous medium, adjustment of stable test concentrations under flow-through conditions was not possible. Therefore, the study was performed under semi-static conditions in 15-L stainless vessels filled with 12 L test medium. Every day (including week-ends), freshly prepared stock solutions were added into the aquaria to adjust the target concentrations. Additionally, to keep good water quality during the study, before addition of the stock solution, every day at least 4 L tempered fresh dilution water was added slowly opposite to the outlet in each aquaria. Hatching and larval survival were recorded everyday throughout the study. At termination of the study (D35), the larvae were weighted blotted dry on a replicate basis. In addition, length was measured in all live larvae at D35.

The concentrations of the test item in the blank control and in one replicate per treatment was measured once a week, before and after addition of the stock solution. Daily exposure was estimated from the geometric mean of the measured concentrations before addition of both fresh dilution water and stock solution and after addition of the stock solution. The biological results were based on the arithmetic mean of the six geometric concentrations estimated for each of the six sampling days. Because no test item could be detected before addition of the stock solution, the limit of detection of 0.2 mg/L was used for calculation of the geometric means, in agreement with the OECD guidance document no. 23 on aquatic toxicity testing of difficult substances and mixtures.

Adverse effects were observed only in the highest concentrated treatment. Hatching rate in the highest treatment (4.6 mg/L nominal ; 0.88 mg/L estimated) was 40% while it was 88% in the control group. Larval survival rate (calculated on the number of hatched larvae) was 48% while it was 86% in the control group. Larval growth (length and weight) remained unaffected in all treatments. NOEC was 2.2 mg/L (nominal), i.e. 0.66 mg/L estimated.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.88 mg/L

Additional information