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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:
2018-02-23 to 2018-03-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 203
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Appearance/Colour: Oily, colourless to light yellow or brownish
Storage Conditions at Test Facility: Approximately 4°C (refrigerator), in the dark, under inert gas
Analytical monitoring:
yes
Details on sampling:
The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director.
One sample from the freshly prepared stock solution and duplicate samples from the freshly prepared test media of the only test concentration and the control were taken at the start of the test and at day 3.
For the determination of the stability of the test item under the test conditions, respectively the maintenance of the test item concentration during the test period, samples were taken in duplicate of the only test concentration and the control at day 1 and at the end of the test (96 hours) from the approximate centre of the aquaria.
All samples were diluted by a factor of 2 with acetonitrile.
Additional samples of the control and the dilution solvent acetonitrile were taken at each sampling without any sample treatment.

All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen (≤ -20 °C) and will be kept stored up to the date of the final report.

-Fortified Samples:
Fortified samples were prepared on the day of analysis. Approximately 50 mg of the test item were dissolved in 50 mL test water to obtain a stock solution of approximately 1 g test item/L. Two independent stock solutions were prepared. Appropriate amounts of these stock solutions were diluted with test water to obtain fortified samples at a level of 2 and 5 mg test item/L. Exact values were documented in the raw data.

-Sample Preparation:
Biological treatment samples and control samples:
The samples were allowed to thaw to room temperature. They were then shaken well and treated with ultrasound for 1 minute to obtain homogenous samples. The samples were already diluted with acetonitrile by factor 2 directly after sampling. The samples were centrifuged (13,000 rpm, 3 minutes) before analysis.
Fortified samples and analytical blank control samples:
The samples were shaken well and were diluted with acetonitrile by factor 2. The samples were centrifuged (13,000 rpm, 3 minutes) before analysis.
Vehicle:
no
Details on test solutions:
-Control: In the control, test water was used without addition of the test item.

-Dosage of Test Item: A stock solution of 30 mg test item/L was prepared by dissolving 33.3, 31.8, 32.2 and 33.7 mg test item into 1110, 1060, 1073.3 and 1123.3 mL test water by intense stirring for 10 minutes. Adequate volumes of this stock solution were diluted with test water to prepare the test media of the desired test concentration.
The test media were prepared just before introduction of the test fish (= start of the test and test medium renewal on day 1, 2 and 3).

-Appearance of the Test Item in Test Medium: There were no remarkable observations.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
-Size and Weight: Juveniles.
The mean body length of the fish* in the test was 4.70 cm ± 0.3 cm (Mean ± SD), the mean body wet weight 1.07 g ± 0.3 g (Mean ± SD).
* 10 fish from the test fish batch were measured before the start of the test
-Sex: Male and female
-Origin: The test fish were obtained from Forellenzuchtbetrieb Störk, 88348 Bad Saulgau, Germany
-Holding Conditions: All fish were obtained and held in the laboratory for at least 12 days before the start of the test. They were held in water of the quality to be used in the test for at least seven days immediately before testing under the following conditions:
-Light: 16 hours photoperiod daily
-Temperature: 13 - 17 °C
-Oxygen concentration: at least 80 % of the air saturation value
-Feeding: three times per week or daily until 24 hours before the test was started
During the last 7 days prior to the start of the test no fish (0 %) died in the test fish batch. Therefore the mortalities in the fish batch were below 5 % and the fish batch was accepted.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
2.5 mmol/L (= 250.0 mg/L) as CaCO3
Test temperature:
13 to 14 °C
pH:
7.6 to 8.1
Dissolved oxygen:
96 to 102 % of the air saturation value
Salinity:
CaCl2 x 2H2O: 2.0 mmol/L (= 294.0 mg/L)
MgSO4 x 7H2O: 0.5 mmol/L (= 123.0 mg/L)
NaHCO3: 0.75 mmol/L (= 65.0 mg/L)
KCl: 0.075 mmol/L (= 5.8 mg/L)
Conductivity:
<=10 µScm^-1
Nominal and measured concentrations:
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of the test item were within ± 20 % of the nominal concentration during the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: 12 litre glass aquaria with 10 litre test medium
- Aeration: The test media were slightly aerated during the test.
Introduction of Fish: At the start of the test 7 fish were introduced into each aquarium in a random order.
Number of Replicates: The test was performed with one replicate per treatment group.
Fish per Replicate: 7
Loading: < 1 g fish/L test water
Exposure Time: 96 hours
Test Procedure: A semi static test with test medium renewal every day was chosen to keep the concentrations of the test item in the test media as constant as possible during the test period of 96 hours. During this semi static test the surviving fish were placed in a clean aquarium with freshly prepared test medium of the corresponding concentration every day. The test media were slightly aerated during the test period.
Feeding: None

Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Sublethal Effects:
In the control and the only test concentration of 3.0 mg test item/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.

96-hour LC50:> 3.0 mg test item/L
95 % Confidence Interval: n.d.
96-hour LC0: 3.0 mg test item/L
96-hour LC100:> 3.0 mg test item/L
96-hour NOEC:≥ 3.0 mg test item/L
96-hour LOEC:> 3.0 mg test item/L
Reported statistics and error estimates:
No statistical analysis was performed.
The LC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.

Observed Mortality of unfed Rainbow Trout exposed to (S)-Nicotine for 96 hours

 Nominal Concentration [mg test item/L]                    Mortality
0 h  2 h   24 h  48 h 
72 h   96 h
 Control  0
 3.0 0  0
 LC50 [mg/L] >3.0  >3.0   >3.0 >3.0   >3.0
 95% Cl n.d   n.d  n.d   n.d   n.d 

# mort: Number of dead fish

n.d.: not determinable

CI.: Confidence interval
Values refer to nominal test concentrations.

Analytical Results

Limit of Detection:

0.2mg test item/L

Limit of Quantification:

1 mg test item/Lafter dilution by factor 2 (corresponding to fortification level of nominal 2 mg test item/L)

105 % (n = 5, RSD 1 %)

Mean Recovery in the Test Samples:


Freshly prepared:      106 % (n = 4, RSD 1 %)

Aged test media:       105 % (n = 4, RSD 2 %)

Validity Criteria of the Analytical Part

Specificity:

No interference of total peak area for the target analyte was found.

Linearity:

Calibration Range:

0.5 – 6mg test item/L

 

Linearity of Response:

Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression

 

Correlation Coefficient:

r = 0.9999

 

Calibration Curve:

y = 133227 * x – 9194

Accuracy and Precision:

Mean Recovery Rates in the Fortified Samples:

104 % (n = 10, RSD 2 %)

The values found for the precision (RSD) and for the accuracy (mean recovery rate) are acceptable.

Conclusion:

The validity criteria for the analytical method have been met.

Validity criteria fulfilled:
yes
Conclusions:
The toxic effect of the test item (S)-Nicotine to Rainbow Trout (Oncorhynchus mykiss) was assessed in a semi static test. Based on the test results the 96-hour LC50 was determined to be > 3.0 mg test item/L based on nominal concentrations. The NOEC was determined to be ≥ 3.0 mg test item/L also based on nominal concentrations.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of the test item were within ± 20 % of the nominal concentration during the test.
Executive summary:

The purpose of this study was to evaluate the acute toxicity of the test item (S)-Nicotine to fish. For this purpose, juvenile Rainbow Trout were exposed in a semi static test to the only concentration under defined conditions for 96 hours. The recorded effects were the mortality and sublethal effects on the fish. A threshold approach was pursued.

The test method of application and the test system are recommended by the test guidelines and Rainbow Trout is one of the recommended test species.

This threshold approach was performed in order to demonstrate that the test item has no toxic effects on the test fish up to the threshold concentration. The lowest EC50value of existing relevant algae or acute invertebrate (e.g. daphnia) tests was set as threshold concentration.

In the interest of animal welfare and efficient use of resources, it is important to avoid the unnecessary use of animals whenever possible. In the field of aquatic toxicology, this especially applies to the acute toxicity testing of fish according to OECD 203. The threshold approach described hereafter addresses fish toxicity by using a single-concentration test (limit test) requiring less fish compared to the full acute fish toxicity study. It is based on the derivation of a threshold concentration from relevant algae and acute invertebrate (e.g. daphnia) tests at which fish toxicity is tested. If no mortality occured in the limit test using the threshold concentration, the threshold concentration might be used as a surrogate LC50value in the further hazard or risk assessment. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

This study encompassed two treatment groups (one test item concentration at nominal 3.0 mg/L and one control) each containing 7 individuals. The acute toxicity to unfed juvenile Rainbow Trout was determined in an aerated, semi-static 96-hour test. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 hours test duration for sublethal effects and mortality.

In the control and the only test concentration of 3.0 mg test item/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time, thus the NOEC was determined to be≥ 3.0 mg test item/L, based on nominal concentrations.

Description of key information

One short-term toxicity study to fish of very high quality (Klimisch 1) is available. The study is GLP-compliant and thus appropriate for risk assessment.

Key value for chemical safety assessment

LC50 for freshwater fish:
3 mg/L

Additional information