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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2014-09-25 to 2014-11-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: A stock solution was first prepared by dissolving 0.020 g test item in 400 mL ISO Medium on day -1. This stock solution was stirred for 20 hours at 300 rpm. The resulted stock solution was the highest test concentration. The highest concentration samples (50 mg/L) were tenfold diluted with water; 6.25, 12.5 and 25 mg/L samples were fivefold diluted with water before the derivatization. The other samples were directly derivatized with FMOC-Cl.

- Stock solution: A stock solution was first prepared by dissolving 0.020 g test item in 400 mL ISO Medium on day -1. This stock solution was stirred for 20 hours at 300 rpm. The resulted stock solution was the highest test concentration.

- Sampling method: Four replicate samples were taken from the test solutions at the start and at the end of the study. From the control one sample was taken at each analytical occasion. The test item hydrolyzes very quickly; therefore concentration of the hydrolysis product was determined. The hydrolysis product, 5-Amino-1,3,3-trimethylcyclohexanemethylamine was derivatized with FMOC-Cl (9-Fluorenylmethyl chloroformate).

- Sample storage conditions before analysis: At room temperature, tightly closed
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: The test solutions used in the test was prepared by mechanical dispersion without using of any solubilising agent. The test solutions were freshly prepared at the beginning of the experiment, in the testing laboratory.

- Test water: Reconstituted water (ISO medium, according to OECD 202 guideline) was used as dilution water in the experiment.

- Differential loading: Separate stock solutions of individual substances were first prepared in deionised water (prepared in TOXI-COOP ZRT. by the reverse osmosis system MILLIPORE, System: Direct Q 5, Serial number: F0DA13956K). The ISO medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.

- Controls:
Untreated control: The dilution water (ISO Medium) was used without addition of the test item.
Toxic reference control: For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Age at study initiation: Less than 24 hours old
- Breeding conditions: Daphnia were bred in the Laboratory of TOXI-COOP ZRT. under similar temperature and light conditions as in the test in Elendt M4-medium (reconstituted water of a similar quality regarding to pH, components of the main ions and total hardness as the test water used in the test).
- Breeding water: Elendt M4-medium
- Acclimatisation: Brood daphnids were acclimatized in ISO medium for 5 days prior to the start of the test.
- Feeding during the test: None

ACCLIMATION
- Acclimation period: 5 days
- Acclimation conditions: Brood daphnids were acclimatized in ISO medium.
- Type and amount of food: The parent animals were fed with centrifuged green alga suspension.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Approx. 249 mg/L (as CaCO3)
Test temperature:
20.5 – 21.0 °C
pH:
In the range of 7.79 – 7.92
Dissolved oxygen:
In the range of 8.22 – 8.58 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 1.563, 3.125, 6.25, 12.5,25 and 50 mg/L.
- Measured concentrations: The measured concentration of the test item in the test solution was in the range of 1.63 – 49.3 mg/L at the start and 1.67 – 52.0 mg/L at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers
- Test volume: Approximately 40 mL test medium
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Volume / Organism: 2 mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202 guideline).
Separate stock solutions of individual substances were first prepared in deionised water (prepared in TOXI-COOP ZRT. by the reverse osmosis system MILLIPORE, System: Direct Q 5, Serial number: F0DA13956K). The ISO medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The water temperature, dissolved oxygen concentration and pH were measured in each test group at the start and in each test vessel at the end of the test. Additionally, the ambient temperature was measured continuously using a min/max thermometer in the climate chamber.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test.
- Photoperiod: The light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness.

EFFECT PARAMETERS MEASURED:
The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: The concentration levels used of the range-finding tests were: Untreated control, 0.1, 1, 10, 50 and 100 mg/L. 10 animals were treated. 5 immobilised animals at 48h were found in a concentrations of 10 mg/L and 10 immobilised animals at 48 h were found in a concentrations of 100 mg/L.
- Test concentrations:
Nominal concentrations: Based on the results of the preliminary tests, the following nominal concentrations were tested in the main study: 1.563; 3.125; 6.25; 12.5; 25 and 50 mg/L (spacing factor for test concentrations: 2.0).
Measured concentrations: The measured concentration of the test item in the test solution was in the range of 1.63 – 49.3 mg/L at the start and 1.67 – 52.0 mg/L at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
8.348 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits (7.362 - 9.465 mg/L))
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.563 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
3.125 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Biological results:
Results after 24 hours: Immobility was observed in the test item concentration range of 3.125 - 50 mg/L after 24 hours of exposure.
Results after 48 hours: In this 48-hour acute toxicity test on Daphnia magna with SIKA Hardener AI (SIKA Härter AI), the 48 h EC50 was 8.348 mg/L and the 48 h EC100 was 50 mg/L. The overall NOEC was determined to be 1.563 mg/L while the overall LOEC was calculated to be 3.125 mg/L (all calculations were based on the nominal concentrations).
Control: The immobilisation rate of the introduced Daphnia did not exceed 10 %. Immobility was not observed in the untreated control and no Daphnia were trapped at the water surface or showed any sign of stress. The dissolved oxygen concentration at the end of the test was more than 3 mg/L in control and test vessels. All validity criteria were fulfilled.
Results with reference substance (positive control):
The 24h EC50 value was determined to be 1.40 mg/L (95 % confidence limits: 1.19 – 1.68 mg/L).
Reported statistics and error estimates:
Statistically not significant immobility (Fisher’s Exact Test (α = 0.05)) was noticed at the test item concentration of 1.563 mg/L. The immobility was statistically significant in the test item concentration range of 3.125 - 50 mg/L (Fisher’s Exact Test (α = 0.05)). Accordingly the NOEC was determined to be 1.563 mg/L and the LOEC was determined as 3.125 mg/L.
The 48-h EC50 was calculated to be 8.348 mg/L (95 % confidence limits: 7.362 – 9.465 mg/L) using Probit analysis by TOXSTAT software
Validity criteria fulfilled:
yes
Conclusions:
In the static immobilization test, young Daphnia magna (< 24 h old) were exposed to the test item for 48 hours according to OECD 202 (2004). The 48 h-EC50 was determined to be 8.3 mg/L and the 48 h-EC100 was determined to be 50 mg/L. The overall NOEC was determined to be 1.6 mg/L while the overall LOEC was calculated to be 3.1 mg/L (all calculations were based on the nominal concentrations).
Executive summary:

In the static immobilization test, young Daphnia magna (< 24 h old) were exposed to the test item for 48 hours according to OECD 202 (2004). The test item hydrolyzes very quickly, therefore concentration of the hydrolysis product was determined. The hydrolysis product, 5-Amino-1,3,3-trimethylcyclohexanemethylamine was derivatized with FMOC-Cl (9-Fluorenylmethyl chloroformate) and than the samples were analyzed by a reverse phase HPLC method using UV detection. The effects are based on the hydrolyzed product. Nevertheless the results can be extrapolated to the test substance itself. Based on the results of the preliminary tests, the following nominal concentrations were tested in the main study: 1.563; 3.125; 6.25; 12.5; 25 and 50 mg/L (spacing factor for test concentrations: 2.0). The measured concentration of the test item in the test solution was in the range of 1.63 – 49.3 mg/L at the start and 1.67 – 52.0 mg/L at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values. The 48 h- EC50 was determined to be 8.3 mg/L and the 48 h-EC100 was determined to be 50 mg/L. The overall NOEC was determined to be 1.6 mg/L while the overall LOEC was calculated to be 3.1 mg/L (all calculations were based on the nominal concentrations).

Description of key information

In a static immobilization test, young Daphnia magna (< 24 h old) were exposed to the test item for 48 hours according to OECD 202 (2004). The 48 h EC50 was determined to be 8.3 mg/L and the NOEC was determined to be 1.6 mg/L (all calculations were based on the nominal concentrations).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
8.3 mg/L

Additional information

In a static immobilization test, young Daphnia magna (< 24 h old) were exposed to the test item for 48 hours according to OECD 202 (2004). The test item hydrolyzes very quickly, therefore concentration of the hydrolysis product was determined. The hydrolysis product, 5-Amino-1,3,3-trimethylcyclohexanemethylamine was derivatized with FMOC-Cl (9-Fluorenylmethyl chloroformate) and than the samples were analyzed by a reverse phase HPLC method using UV detection. The effects are based on the hydrolyzed product. Nevertheless the results can be extrapolated to the test substance itself. Based on the results of the preliminary tests, the following nominal concentrations were tested in the main study: 1.563; 3.125; 6.25; 12.5; 25 and 50 mg/L (spacing factor for test concentrations: 2.0). The measured concentration of the test item in the test solution was in the range of 1.63 – 49.3 mg/L at the start and 1.67 – 52.0 mg/L at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values. The 48 h EC50 was determined to be 8.3 mg/L and the 48 h EC100 was determined to be 50 mg/L. The overall NOEC was determined to be 1.6 mg/L while the overall LOEC was calculated to be 3.1 mg/L (all calculations were based on the nominal concentrations).