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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:
18 April 2018 - 17 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Storage Conditions at Test Facility: At 20 ± 5 °C under Nitrogen, in the dark
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentrations of the test item were analysed in all of the duplicate test media samples from all test concentrations and the control from both sampling times (0 and 48 hours). Since stand-by analysis was performed, test start (analysis 1) and test end samples (analysis 2) were analysed in two separate analyses.
- Sampling method: The sample s 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. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the controls were taken at the start of the test. 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 test period, duplicate samples from the test media of all test concentrations and the controls were taken at the end of the test (after the 48 hours test period) by pooling the contents of the test beakers of each treatment. The samples remained undiluted until sample preparation which was performed stand-by directly after sampling.
- Sample storage conditions before analysis: All samples were analysed stand-by immediately after sampling via Headspace-extraction followed by GC-MS analysis. Therefore, sample storage was not feasible.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 137 μL test item into 1000 mL test water by intense stirring for 15 minutes. The pH of the stock solution was adapted with 2mol HCl from 10.5 to 8.3.Adequate volumes of this stock solution were diluted with test water to prepare the test media. The test media were prepared just before introduction of the daphnids (= start of the test). The appearance of the test item in the test media was observed at the start of the test and after 24 and 48 hours test duration in all test concentrations.
- Controls: Test water -> reconstituted Water (Elendt "M4")
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5, Female
- Source: The daphnids introduced in the test were taken from our in-house laboratory culture.
- Age at Test Start: From 1.75 to 23.5 hours old
The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in the same reconstituted water as the test water used in the test. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon. For the evaluation of the quality of the daphnids clone used and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

ACCLIMATION
- Acclimation period: Was not necessary, since the test was performed in the same medium as the culturing
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
2.5 mmol/L (= 250 mg/L) as CaCO3
Test temperature:
20.4 to 20.9 °C at test start;
20.1 to 20.3 °C at test end
pH:
7.9 to 8.4 at test start;
7.9 to 8.1 at test end and thus the pH-value did not vary by more than 1.5 units
Dissolved oxygen:
8.8 to 9.1 mg/L at test start;
8.7 to 8.8 mg/L at test end
Conductivity:
< 5 μScm-1
Nominal and measured concentrations:
100, 45.5, 20.7, 9.4 and 4.3 mg test item/L and a control corresponding to the following geometric mean measured concentrations: 85.0, 37.8, 17.6, 9.20 and 3.93 mg test item/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers of 110 mL volume containing as much test medium as possible (i.e. the remaining head space was reduced to a technical possible minimum of some mL). Each test unit was uniquely identified with serial number, treatment and replicate number.
- Type (delete if not applicable): The vessels were kept closed during the whole period of the study with a conical glass stopper to avoid loss of the test item due to volatilisation.
- No. of organisms per vessel: 20 daphnids per control and test concentration, divided into 4 groups of 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
(Elendt "M4"): Analytical grade salts and additives were added at the following nominal concentrations in deionised water (conductivity < 5 μScm-1):
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·2H2O 2.5 mg/L
FeSO4·7 H2O 1.0 mg/L

Vitamins
Thiamin HCl 75.0 μg/L
Cyanocobalamin (B12) 1.0 μg/L
Biotin (B6) 0.75 μg/L

Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3 Alkalinity: 0.9 mmol/L

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the stock solution was adapted with 2 mol HCl from 10--5 to 8.3
- Light intensity: 560 to 770 Lux (measured once during the test)


RANGE-FINDING STUDY
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions. The results of the pre-test are given in Appendix 2.
The pre-experiments were not performed in compliance with the GLP-Regulations and will be 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.

Table 1: Results of the non-GLP Range-finding Test

Test item concentration [mg/L] No. of daphnids tested No. Immobilised % immobilisation
24 h 48 h 24 h 48 h
Control 10 0 0 0 0
0.1 10 0 0 0 0
1 10 0 0 0 0
10 10 0 0 1 10
100 10 1 10 2 20
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
>= 85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
LOEC
Effect conc.:
> 85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
> 85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Control Immobilisation Rate: Should not exceed 10 %. Furthermore, not more than 10 % of the daphnids should show signs of disease or stress or unusual behaviour such as trapping at surface of water. -> Was 0 % and furthermore no daphnids showed signs of disease or stress; thus the validity criterion was met.
Dissolved Oxygen Concentration: At the end of the test the dissolved oxygen concentration in the test media should be ≥ 3 mg O2/L in the control and test vessels. -> Was ≥ 8.7 mg O2/L in the control and test vessels at the end of the test; thus validity criterion was met.
- Signs of Intoxication after 24 Hours: After 24 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 37.8 mg test item/L. At the concentration of 85.0 mg test item/L, one animal of 20 was immobile. This immobility was statistically not significant (Fisher’s Exact Binominal Test with Bonferoni Correction; α = 0.05, one-sided greater).
- Signs of Intoxication after 48 Hours: 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 17.6 mg test item/L. At the concentration of 37.8 and 85.0 mg test item/L, one animal of 20 was immobile. This immobility was statistically not significant (Fisher’s Exact Binominal Test with Bonferoni Correction; α = 0.05, one-sided greater).
Appearance of the Test Item in the Test Media: There were no remarkable observations in all test concentrations or the control.
Reported statistics and error estimates:
At the start of the test the recoveries of the test item ranged from 84 - 122 % of the nominal test concentrations. After 48 hours test duration, the recoveries ranged from 65 - 85 % of the nominal values. Since not all test concentrations remained within ± 20% of the initial measured concentration, all biological endpoints were calculated based on geometric mean concentrations.
Validity criteria fulfilled:
yes
Conclusions:
The toxic effect of the test item to Daphnia magna was assessed in a static dose-response test; all endpoints are based on based on geometric mean concentrations. The 48-hour NOEC was determined to be ≥ 85.0 mg test item/L. The 48-hour LOEC was determined to be > 85.0 mg test item/L and the 48-hour EC10,20,50 values were also determined to be > 85.0 mg test item/L
Executive summary:

1.1 Study Design

The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna. Young daphnids (< 24 hours old) were exposed to the test item added to test water at a range of concentrations in a static test for 48 hours. Twenty test organisms per concentration were used (four replicates with 5 daphnids each). The Daphnia were exposed to the following nominal test concentrations 100, 45.5, 20.7, 9.4 and 4.3 mg/L, along with a dilution water control. The study was conducted according to OECD 202 (2004).

Endpoints reported are the EC10,20,50 (24 and 48 h) values and the NOEC (No Observed Effect Concentration). Temperature, pH-value and oxygen concentration of the test solutions measured after 0, 24 and 48 hours are also reported.

1.2 Results

The analytical verification of concentrations in daphnid test medium was done by analysing the content of test item in the samples taken from the test vessels at 0 and 48 hours. At the start of the test the recoveries of the test item ranged from 84 - 122 % of the nominal test concentrations. After 48 hours test duration, the recoveries ranged from 65 - 85 % of the nominal values. Since not all test concentrations remained within ± 20% of the initial measured concentration, all biological endpoints were calculated based on geometric mean concentrations. 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 17.6 mg test item/L. At the concentration of 37.8 and 85.0 mg test item/L, one animal of 20 was immobile.

The toxicological sensitivity of the daphnids at the testing facility is confirmed twice annually using five concentrations of potassium dichromate. The results of the most recent test indicate an EC50 (24 h) of the reference item potassium dichromate of 1.06 mg test item/L. Since the results fall within the range mentioned in guideline OECD 202 and the historical data generated with the reference item at the testing facility, the daphnids were suitable for the determination of the toxicological effects of the test item.

1.3 Conclusion

The toxic effect of the test item to Daphnia magna was assessed in a static dose-response test; all endpoints are based on based on geometric mean concentrations. The 48-hour NOEC was determined to be ≥ 85.0 mg test item/L. The 48-hour LOEC was determined to be > 85.0 mg test item/L and the 48-hour EC10,20,50 values were also determined to be > 85.0 mg test item/L

Description of key information

The acute toxicity of Isobutylamine to Daphnia magna was tested in a static 48 -hour Immobilisation GLP test with closed vessel design according to OECD 202 guideline. The 48-hour NOEC was determined to be ≥ 85.0 mg test item/L. The 48-hour LOEC was determined to be > 85.0 mg test item/L and the 48-hour EC10,20,50 values were also determined to be > 85.0 mg test item/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
85 mg/L

Additional information