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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:
2010-05-03 to 2010-05-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test item concentrations and the control were analytically verified at the beginning and end of the test.
- Sampling method: The highest test item concentration of nominal 16 mg/L was analysed after 48 h, due to 100 % mortality.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Eluate: Dilution water
- Differential loading: 1 - 2 - 4 - 8 - 16 mg/L
- Controls: 7 fish in dilution water without test item were tested under the same test conditions as the test replicates.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, Hildesheim, Germany
- Length at study initiation (length definition, mean, range and SD): Average body length 1.76 cm
- Weight at study initiation (mean and range, SD): Average body weight 0.06 g
- Method of breeding: Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons x m-2 x s-1, natural photoperiod). Tap water of local origin was used for holding and testing. The water was filterred on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters: Total hardness: 10 - 250 mg CaCO3/L; pH-value: 6.0 - 8.5. The water is analysed biannula acc. to German tap water regulation. The water was changed at least once per week. the dissolved oxygen concentration was more than 80 % of the air saturation value.
- Feeding during test: No feeding during test



ACCLIMATION
- Acclimation period: Zebrafish with al least 12 days of acclimatisation and mortality < 5% within these days before the study starts were used.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Stoer perlets, SERA GmbH, 52518 Heidelberg, Germany
- Feeding frequency: 3 times per week
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered thwoughout holding and testing.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Total Hardness at day 0: 56 mg/L as CaCO3
Test temperature:
Mean measured test item concentration [mg/L] Temperature
[°C]
Day 0 14.6 23.7
6.56 23.8
3.43 23.8
1.59 23.8
0.734 23.7
Control 23.6
Day 1 14.6 24.2
6.56 24.2
3.43 24.2
1.59 24.1
0.734 24.1
Control 24.2
Day 2 14.6 24.1
6.56 24.2
3.43 24.2
1.59 240.
0.734 23.8
Control 23.6
Day 3 14.6 -
6.56 24.0
3.43 24.0
1.59 24.1
0.734 23.7
Control 23.4
Day 4 14.6 -
6.56 24.3
3.43 24.2
1.59 24.3
0.734 24.2
Control 23.6
pH:
Mean measured test item concentration [mg/L] pH Value
Day 0 14.6 7.34
6.56 7.31
3.43 7.29
1.59 7.19
0.734 7.17
Control 7.15
Day 1 14.6 7.60
6.56 7.60
3.43 7.58
1.59 7.56
0.734 7.55
Control 7.53
Day 2 14.6 7.54
6.56 7.54
3.43 7.51
1.59 7.55
0.734 7.28
Control 7.13
Day 3 14.6 -
6.56 7.30
3.43 7.31
1.59 7.30
0.734 7.27
Control 7.02
Day 4 14.6 -
6.56 7.46
3.43 7.45
1.59 7.41
0.734 7.38
Control 7.11
Dissolved oxygen:
Mean measured test item concentration [mg/L] Oxygen- Saturation
[%]
Day 0 14.6 100
6.56 100
3.43 100
1.59 100
0.734 100
Control 100
Day 1 14.6 99
6.56 99
3.43 99
1.59 100
0.734 100
Control 100
Day 2 14.6 97
6.56 97
3.43 99
1.59 100
0.734 100
Control 100
Day 3 14.6 -
6.56 88
3.43 95
1.59 97
0.734 99
Control 100
Day 4 14.6 -
6.56 92
3.43 95
1.59 97
0.734 98
Control 100
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods"
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: glass, 2 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): One application at test start
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water, local origin, pH: 6.0 - 8.5
- Total organic carbon: 10 - 250 mg CaCO3/L
- Chlorine: The dilution water was filtered as described above
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water is analysed biannual


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: natural
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: Yes
Cumulative Mortality [%] in the Preliminary
(n = 5)
Nominal test item concentration
[mg/L] Test Duration [hours]
24 48 72 96
10 0 80 100 ---
1 0 0 0 0
Control 0 0 0 0

Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.59 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
5.74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not observed
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
LC50-values and 95 % confidence intervals after 24, 72 and 96 hours exposure were calculated by sigmoidal dose-response regression. After 48 hours only 0 and 100 % mortality were found. No evaluation via regression analysis was carried out.
Hence the LC50 is the mean measured value of the concentrations with 0 and 100 % mortality. The concentration causing 0 and 100 % mortality were chosen as lower and upper confidence limit.
The concentrations causing 0 and 100 % mortality (LC0 and LC100) as well as the no effect level (NOEC) after 96 h were carried out using standard procedures.
Sublethal observations / clinical signs:

Observations in the Test Vessels

Mean measured test item concentration [mg/L]

Effect*

Test Duration [h]

2

24

48

72

96

14.6

(E)

-

6/7

1/1

---

---

(2.3)

5/7

1/7

-

---

---

(1)

2/7

-

-

---

---

   6.56

(E)

-

-

-

3/7

2/4

(2.6)

-

-

-

1/7

-

(2.3)

3/7

7/7

7/7

3/7

2/4

(1)

4/7

-

-

-

-

   3.43

(2.3)

-

-

-

2/7

2/7

(1)

7/7

7/7

7/7

5/7

5/7

   1.59

(1)

7/7

7/7

7/7

7/7

7/7

    0.734

(1)

7/7

7/7

7/7

7/7

7/7

Control

(1)

7/7

7/7

7/7

7/7

7/7

Cumulative Mortality [%] in the Test Vessels

Mean measured test item concentration

[mg/L]

Test Duration [h]

2

24

48

72

96

14.6

0

86

100

100

100

   6.56

0

0

0

43

71

   3.43

0

0

0

0

0

   1.59

0

0

0

0

0

    0.734

0

0

0

0

0

Control

0

0

0

0

0

              

Validity criteria fulfilled:
yes
Conclusions:
In this study the test item was found to cause toxic effects to Zebrafish after 96 hours at a mean measured test item concentration of ≥ 3.43 mg/L (LOEC).
The nominal test item concentration LC50 (96 hours) was 5.74 (5.55 – 5.94) mg/L.
The NOEC after 96 hours was 1.59 mg/L (mean measured test item concentration).
Executive summary:

The acute toxicity of the test item to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992). A static test with 5 test concentrations of nominal 1 – 2 – 4 – 8 - 16 mg/L (factor 2) was performed, corresponding to mean measured concentrations of 0.734 – 1.59 – 3.43 – 6.56 – 14.6 mg/L.

Duration of the test was 96 h. Seven test organisms were exposed to each test concentration and control. Water quality parameters pH-value, temperature and oxygen-saturation measured after 0, 2, 24, 48, 72 and 96 h were determined to be within the acceptable limits.

The concentrations of the test item were analysed via LC-MS/MS at all concentration levels at test start and test end or whenever 100 % mortality occurred. Recovery rates of the test item at the beginning of the study were in the range of 94 - 101 % and at the end in the range of 53 – 84 % of the nominal values. For details of the analytical results please refer to part 9. All effect levels are given based on mean measured concentrations of the test item.

LC-Values with 95 % Confidence Intervals

                                               (based on mean measured test item concentrations)

Test duration [h]

LC 50

p = 95 % [mg/L]

24

11.1

10.6 – 11.7

48

10.6

6.56 – 14.6 *

72

6.85

6.78 – 6.93

96

5.74

5.55 – 5.94

LC100 =

14.6

Lowest test item concentration

with 100 % mortality after 96 h

LC0 =

3.43

Highest test item concentration

with 0 % mortality after 96 h

NOEC (0-96 h) =

No observed effect concentration

1.59

Description of key information

Short term toxicity to fish, OECD  203 , GLP, LC50: 5.74 mg/l, 96h - (PARAD consortium, NOACK, 2010, D. Scheerbaum) key study

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
5.74 mg/L

Additional information

Short term toxicity to fish - OECD 203 – LC50: 5.74 mg/l, 96h - (PARAD consortium, NOACK, 2010, D. Scheerbaum)

The acute toxicity of the registered substance (test item) to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992). A static test with 5 test concentrations of nominal 1 – 2 – 4 – 8 - 16 mg/L (factor 2) was performed, corresponding to mean measured concentrations of 0.734 – 1.59 – 3.43 – 6.56 – 14.6 mg/L. Duration of the test was 96 h. Seven test organisms were exposed to each test concentration and control. Water quality parameters pH-value, temperature and oxygen-saturation measured after 0, 2, 24, 48, 72 and 96 h were determined to be within the acceptable limits. The concentrations of the test item were analysed via LC-MS/MS at all concentration levels at test start and test end or whenever 100 % mortality occurred. Recovery rates of the test item at the beginning of the study were in the range of 94 - 101 % and at the end in the range of 53 – 84 % of the nominal values. For details of the analytical results please refer to part 9. All effect levels are given based on mean measured concentrations of the test item. After 96h an LC50: = 5.74 mg/l was determined.

The test item is toxic for fish.

Evaluation: valid without restrictions