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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 2018 - Feb 2018
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
Only 19 daphnids, divided into 4 groups of 5 or 4 animals were introduced in the lowest concentration of 0.43 mg test item/L
Principles of method if other than guideline:
Only 19 daphnids, divided into 4 groups of 5 or 4 animals were introduced in the lowest concentration of 0.43 mg test item/L, instead of 20, due to human error. There was no effect on the study because the NOEC was observed to be at 2.1 mg test item/L and all validity criteria were fulfilled.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
At least one sample from the freshly prepared stock solution (if prepared) and at least duplicate samples from the freshly prepared test media of all test concentrations and the control will be taken at test start and the test medium renewal after 24 hours.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the renewal periods, at least duplicate samples will be taken at the test medium renewal and at the end of the test from all aged test media and the control of the actual test by pooling the contents of the beakers of all replicates for each treatment.
All samples will be diluted by a factor of 2 with acetonitrile.
Additional samples of the control blank and the dilution solvent, if used, will be taken at test start and test end without any sample treatment.
All samples will be stored in a freezer (≤ -20 °C) immediately after sampling, and will be kept stored until analysis, unless analysis is performed directly after sampling. Spare samples will be stored in a freezer until delivery of the final report to enable additional analyses to be conducted on request of the Sponsor. After delivery of the final report, all samples will be discarded. If in a pre-experiment for investigation of the storage stability a significant loss of test item is determined in freezer stored test medium samples, all samples will be analysed immediately after sampling without prior storage.
The concentrations of the test item (S)-Nicotine will be measured in all duplicate test medium samples. However, the samples from test concentrations below the 48-hour NOEC and from the stock solution need not to be analysed. From the control samples at least one of the duplicate samples will be analysed from each sampling date. The additional samples of the control blank and the dilution solvent will only be analysed if necessary.
Details on test solutions:
Pre-Experiments: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions. The pre-experiments were not performed in compliance with the GLP-Regulations and were excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the project number of the present study.

Test Concentrations: 10, 4.5, 2.1, 0.94 and 0.43 mg test item/L and a control.

Control: In the control, test water was used without addition of the test item.

Dosage of Test Item: A stock solution of 40 mg test item/L was prepared by dissolving 21.4 and 22.2 mg test item into 535 and 555 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 concentrations. The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start: Less than 24 hours old
Sex: Female
Origin: The daphnids introduced in the test will be taken from ibacon's in-house laboratory culture.
Breeding Conditions: The daphnids are bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids is performed in Elendt M4 medium. The test organisms will not be first brood progeny. The daphnids in the stock culture are fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Reference Item: For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Acclimatisation: Is not necessary since the breeding is performed in the same water.

Introduction of Daphnids: 20 daphnids per control and test concentration, divided into 4 groups of 5 animals, each group in 60 mL test medium
Replicates: The test was performed with four replicates per treatment group.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
2.5 mmol/L (=250 mg/L) as CaCO3
Test temperature:
20.2 to 20.7 °C in the freshly prepared media;
19.2 to 19.6°C in the aged test media
8.1 to 8.4 in the freshly prepared media;
7.8 to 8.0 in the aged test media; and thus the pH-value did not vary by more than 1.5 units
Dissolved oxygen:
8.9 to 9.1 mg/L in the freshly prepared media;
8.7 to 8.9 mg/L in the aged test media
Elendt "M4"
Main Compounds

CaCl2•2 H2O: 293.80 mg/L
MgSO4•7 H2O: 123.30 mg/L
KCl: 5.80 mg/L
NaHCO3: 64.80 mg/L
Na2SiO3•9 H2O: 10.00 mg/L
NaNO3: 0.27 mg/L
KH2PO4: 0.14 mg/L
K2HPO4: 0.18 mg/L

Trace Elements
H3BO3: 2860 µg/L
MnCl2•4 H2O: 361 µg/L
LiCl: 306 µg/L
RbCl: 71 µg/L
SrCl2•6 H2O: 152 µg/L
NaBr: 16 µg/L
Na2MoO4•2 H2O: 63 µg/L
CuCl2•2 H2O: 17 µg/L
ZnCl2: 13 µg/L
CoCl2•6 H2O: 10 µg/L
KI: 3.3 µg/L
Na2SeO3: 2.2 µg/L
NH4VO3: 0.6 µg/L
Na2EDTA•2 HO: 2.5 mg/L
FeSO4•7 H2O: 1.0 mg/L

Thiamin HCl: 75.0 µg/L
Cyanocobalamin (B12): 1.0 µg/L
Biotin (B6): 0.75 µg/L
< 5 µScm^-1
Nominal and measured concentrations:
10, 4.5, 2.1, 0.94 and 0.43 mg test item/L and a control.
Details on test conditions:
The test was performed in the dark.
Test conditions were recorded with suitable instruments and documented in the raw data.
Glass beakers of 100 mL volume containing approximately 60 mL of test medium. The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
Each test unit was uniquely identified with serial number, treatment and replicate number.
A semi-static test system with a test medium renewal on Day 1 was chosen to keep the concentrations of test item as constant as possible in the test media.
Reference substance (positive control):
Potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
3 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CI = 1.68 - 5.36 mg/L
48 h
Dose descriptor:
Effect conc.:
2.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
4.5 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
24 h
Dose descriptor:
Effect conc.:
3.75 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
95% CI = < 0.43 - > 10
Details on results:
After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 2.1 mg test item/L. At the two highest concentrations of 4.5 and 10 mg test item/L, all animals were immobile.
Results with reference substance (positive control):
Results of the most recent Test with the Reference Item Potassium dichromate performed in June 2017 (88298220):

After 48 h:
EC50 [mg/L]: 1.07
95 % CI [mg/L]:1.34 - 1.85 n.d.
EC20 [mg/L]: 0.983
95 % CI [mg/L]:n.d.
EC10 [mg/L]:0.938
95 % CI [mg/L]:n.d.
NOEC [mg/L]: 0.5
LOEC [mg/L]: 1.0

Values refer to nominal test concentrations
CI: Confidence interval
n.d.: not determinable
NOEC and LOEC were determined directly from the raw data.
Reported statistics and error estimates:
The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by logit analysis.
The NOEC and LOEC after 24 and 48 hours were determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Analytical results

Sample Description [mg test item/L] % of nominal*   RSD [%]
Control <LOD  n.a. 
0.43 98 
0.94  103 
2.1  98 
4.5  98 
10  101 

*mean value of all measured samples per treatment group
RSD: relative standard deviation per treatment group
n: number of analysed samples; n.a.: not applicable

Influence of (S)-Nicotine on the Mobility of Daphnia magna

 Nominal Concentration [mg test item/L]     No. of Daphnia tested    Number of immobilised Daphnia after      % of immobilised Daphnia after   
 24h 48h  24h  48h 
 Control 20  0  0  0  0
 0.43 19  0
 0.94 20
 2.1 20 0 0 0
 4.5 20 20 20 100  100


18 20 90  100 
Validity criteria fulfilled:
The toxic effect of the test item (S)-Nicotine to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be 2.1 mg test item/L. The 48-hour LOEC was determined to be 4.5 mg test item/L and the 48-hour EC50 value was calculated to be 3.00 mg test item/L.
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 ± 10 % of the nominal concentrations during the test.
Executive summary:

The purpose of this study was to determine the influence of the test item (S)-Nicotine on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a semi-static test tovarious concentrations under defined conditions for 48 hours. The recorded parameters were the effects on the mobility of the daphnids after 24 and 48 hours. The test method of application and the test system are recommended by the test guidelines stated below and Daphnia magna is the recommended test species.

The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Test guidelines:

- Commission Regulation (EC) No 440/2008, Annex, Part C, C.2.: "Daphniasp. Acute Immobilisation Test", Official Journal of the European Union (EN), dated May 30, 2008

- OECD Guideline for Testing of Chemicals No. 202: "Daphniasp., Acute Immobilisation Test" adopted April 13, 2004

- SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Description of key information

Classification for short-term toxicity to aquatic invertebrates is based on a key study of high quality (Klimisch1). The study is GLP compliant and appropriate for risk assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
3 mg/L

Additional information