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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Remarks:
Threshold approach according to OECD 126
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-01 to 2016-08-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.30 mg/L was tested as a threshold concentration in a limit test.

- Sampling method:
The limit concentration and the control were analytically verified via LC-MS/MS from freshly prepared media after 0 and 72 h and from corresponding aged test media after 24 and 96 h.



Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1000 mg/L (total volume 1000 mL) was prepared in demineralised water. A suitable glass Schott bottle with a stirring rod was placed on a balance and was tarred. 1000 mg of the test item were weighed into this glass bottle. Approximately 500 mL demineralised water was added. This solution was acidified with HCl (2 M, pH-value not below 3) until the solution had become milky. At this point the solution was stirred with 1100 rpm. The pH value was held at 3-4 by adding HCl (2 M). When this emulsion was homogenous, the solution was neutralized to pH 5-6. Finally, the emulsion was filled up with demineralised water to the desired weight (to achieve 1000 mg/L). This solution was used to prepare the nominal test item concentration 0.30 mg/L. This procedure was repeated daily for preparation of the stock solution for the daily water renewal.

- Eluate: Dilution water
- Differential loading: 0.30 mg/L was tested as a threshold concentration in a limit test.

The concentrations are based on the results of a non-GLP preliminary test carried out under static conditions.

- 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: All fish used in the test were gained at NOACK LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Length at study end (length definition, mean, range and SD): Average body length: 2.66 cm
- Weight at study end (mean and range, SD): Average body weight: 0.204 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test


ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- 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: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.


Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
Total Hardness at day 0: 61 mg/L
Test temperature:
Please refer to "Any other information on materials and methods"
pH:
Please refer to "Any other information on materials and methods"
Dissolved oxygen:
Please refer to "Any other information on materials and methods"
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods incl. tables"
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 aquaria of 13 L were used, filled with approximately 10 L (dimensions: 25.5/18/28 cm) and loosely covered with glass
- Aeration: The test vessels were gently aerated during exposure.
- Renewal rate of test solution (frequency/flow rate): A semi-static test with daily renewal of the test media was performed.
- Control: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates
- Reference item: No reference item is recommended for this test according to the guideline.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control and pH-control (replicates): 1
- Biomass loading rate: 0.143 g fish per L test solution


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 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Alkalinity: 0.60 mmol/L (measured quarterly)
Acidity: 0.10 mmol/L (measured quarterly)
Conductivity: 174 µS/cm (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 every 24 hours.
Total hardness of the water was determined at the beginning out of the control. During the test the water temperature was recorded continuously once per hour with a data logger. The light intensity on the surface of the test aquaria was measured at the start of the exposure.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A photoperiod of 15 h light / 9 h dark corresponding to natural daylight hours occurred during the course of the study.
- Light intensity: 7 - 750 Lux


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 2, 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: None







Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
0.272 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: not applicable
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.272 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: not applicable
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 0.272 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: not applicable
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): The test solution was visually clearly dissolved throughout the period of exposure.
Results with reference substance (positive control):
No reference substance tested
Reported statistics and error estimates:
Throughout the exposure no mortality occurred. Therefore, the LC-values were not calculated but determined directly from the test results.

After 96 hours, 0 % mortality occurred in both the control and the limit concentration. Hence, the LC0 , LC50 and LC100 were determined from these raw data. The NOEC and LOEC were determined from the recorded observations without any statistical calculations.


The concentrations of the test item TETRAMEEN 2 HT were determined at the start of the exposure intervals (0 h and 72 h) and at the end of the exposure intervals (24 h and 96 h) of the tested level and the control via LC-MS/MS.

The measured concentrations of the test item were 98 and 117 % of the nominal concentration at the start of the exposure intervals. The concentrations decreased to 74 and 76 % at the end of the exposure intervals. Therefore the geometric mean measured test item concentration was calculated to be 0.272 mg/L.

Observations in the Test Vessels

(n = 7)

Geometric mean measured test item concentration
[mg/L]

Effect *)



Number of fish effected
after different exposure periods [hours

2

24

48

72

96

0.272

(1)

7/7

7/7

7/7

7/7

7/7

Control

(1)

7/7

7/7

7/7

7/7

7/7

*) The numbers in brackets correspond to the following observations: 

(1)          = Normal behaviour

Cumulative Mortality [%] in the Test Vessels

(n = 7)

Geometric mean measured test item concentration
[mg/L]

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

2

24

48

72

96

0.272

0

0

0

0

0

Control

0

0

0

0

0

Validity criteria fulfilled:
yes
Conclusions:
All effect levels are given based on the geometric mean measured concentration of the test item TETRAMEEN 2HT. The limit concentration of the test item was found to have no effect on the vitality of zebrafish. In conclusion, the LC0 corresponds to the geometric mean measured limit concentration 0.272 mg/L. The LC50 and the LC100 correspond to the geometric mean measured limit concentration > 0.272 mg/L.





Executive summary:

The acute toxicity of the test item Tetramine di-C16 -18 (batch no.:890000394200) to fish (zebrafish) was determined according to the OECD Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation No. 440/2008/C.1 (2008) at the test facility from 2016-08-01 to 2016-08-07 with a definitive exposure phase from 2016-08-01 to 2016-08-05.

 

A semi static test was performed as a limit test with a concentration of 0.30 mg/L, corresponding to the geometric mean measured test item concentration 0.272 mg/L. The test had a duration of 96 hours. Seven test organisms were exposed each for the test concentration and the control. The water quality parameters pH-value, temperature and oxygen saturation, measured after 0, 24, 48, 72 and 96 h, were determined to be within the acceptable limits.

 

Analytical evaluation of limit test item concentration of Tetramine di-C16 -18 and the control were carried out from freshly prepared media after 0 and 72 hours and from corresponding aged test media after 24 and 96 hours by LC-MS/MS.The measured concentrations of Tetramine di-C16 -18 in the fresh media (0 and 72 h) were 98 and 117% of the nominal values. The measured concentrations in the old media (24 and 96 h) were 74 and 78% of the nominal values. Therefore, all effect values given are based on the geometric mean measured test item concentrations of Tetramine diC16 -18.

The results are summarized in the following table:

 

EC-Values (0 – 96 hours) of the Test Item

Based on the geometric mean measured concentration of the test item [mg/L]

Test duration [h]

LC50

Confidence interval
p = 95 %

2

> 0.272

n.a.

24

> 0.272

n.a.

48

> 0.272

n.a.

72

> 0.272

n.a.

96

> 0.272

n.a.

LC100=

> 0.272

Lowest test item concentration

with 100% mortality after 96 h

LC0=

0.272

Highest test item concentration

with 0% mortality after 96 h

           n.a. = not applicable

 

 

Description of key information

For alkylamines in general fish are less sensitive when compared to daphnia and algae. This difference is however in most cases less than a factor of 10.

For tetramine di-C16 -18 an LC50 (96 h, semi-static) has been observed of >0.272 mg/L based geometric mean measured concentrations. The study has been performed as a limit test according to OECD 126 threshold approach using the observe acute algae and daphnia EC50 values as reference points.

Key value for chemical safety assessment

LC50 for freshwater fish:
0.272 mg/L

Additional information

The acute toxicity of the test item tetramine di-C16 -18 (batch no.:890000394200) to fish (zebrafish) was determined according to the OECD Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation No. 440/2008/C.1 (2008) at the test facility from 2016-08-01 to 2016-08-07 with a definitive exposure phase from 2016-08-01 to 2016-08-05.

 

A semi static test was performed as a limit test with a concentration of 0.30 mg/L, corresponding to the geometric mean measured test item concentration 0.272 mg/L. The test had a duration of 96 hours. Seven test organisms were exposed each for the test concentration and the control. The water quality parameters pH-value, temperature and oxygen saturation, measured after 0, 24, 48, 72 and 96 h, were determined to be within the acceptable limits.

 

Analytical evaluation of limit test item concentration of Tetramine diC16 -18 and the control were carried out from freshly prepared media after 0 and 72 hours and from corresponding aged test media after 24 and 96 hours by LC-MS/MS.The measured concentrations of Tetramine di-C16 -18 in the fresh media (0 and 72 h) were 98 and 117% of the nominal values. The measured concentrations in the old media (24 and 96 h) were 74 and 78% of the nominal values. Therefore, all effect values given are based on the geometric mean measured test item concentrations of Tetramine di-C16 -18.

The limit concentration of the test item was found to have no effect on the vitality of zebrafish. In conclusion, the LC0 corresponds to the geometric mean measured limit concentration 0.272 mg/L. The LC50 and the LC100 correspond to the geometric mean measured limit concentration > 0.272 mg/L.