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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:
16 August 2017 to 18 August 2017
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:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF 2-7-2-1
Version / remarks:
Ref. No. 12-Nousan-8147 on 24 November 2000 & Ref. No.13-Seisan- 3986 on 10 October 2001.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Analytical measurements were performed from the control and at the applied test concentration levels at the beginning and at the end of the experiment.
Vehicle:
no
Details on test solutions:
- A stock solution with a concentration of 100.0 mg/L (nominal) was prepared with direct addition of the test material, mixed into the test medium (ISO Medium) using ultrasonic bath (approximately 40 minutes). The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of the treatments).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Breeding: The Daphnia are bred at the testing laboratory. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
- Age: Less than 24 h old at the beginning of the test.
- Feeding during test: No

ACCLIMATION
- Acclimation period: There was no acclimatisation because the water used was similar to the culture water.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The reconstituted water (ISO medium) had a total hardness of 247 mg/L (as CaCO3). The dilution water (1:1 v/v solution of ISO medium and deionised water) used in the experiment had a total hardness of 118 and 121 mg/L (as CaCO3), respectively at the start of the treatments.
Test temperature:
20.4 to 20.8 °C
pH:
7.11 to 7.75
Dissolved oxygen:
7.7 to 8.6 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
- Mean measured concentrations: 6.71, 13.30, 26.75, 53.04 and 104.12 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers
- Material, size, headspace, fill volume: Glass beakers with at least 5 mL test solution / animal.
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per vehicle control: 4

TEST MEDIUM / WATER PARAMETERS
- Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition was used for breeding the test animals.
- Composition of ISO medium: Separate stock solutions of individual trace elements were first prepared in deionised water at the concentrations below. The medium was prepared of these different stock solutions, so it contained all trace elements (combined solution). 25 mL/L of each stock solution was added to deionised water to prepare the media.
- CaCl2.2H2O: 11.76 g/L
- MgSO4.7H2O: 4.93 g/L
- KCl: 0.23 g/L
- NaHCO3: 2.59 g/L

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. The pH was measured at
the start and at the end of the renewal periods in each test vessel and was in the range of 7.11 – 7.75.
- Photoperiod: The test was carried out in 16-hour light and 8-hour dark cycle.
- Temperature: The water temperature was measured at the start of the experiment and in 24-hour intervals thereafter in each test vessel. The test temperature was in the range of 20.4 – 20.8 °C measured in the test vessels. The additionally measured temperature in the climate chamber was between 20.1 and 21.0 °C.
- Oxygen concentration: The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the renewal periods and was in the range of 7.7 – 8.6 mg/L.

EFFECT PARAMETERS MEASURED:
- The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
- The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.
- The water temperature, the oxygen concentrations and pH of the controls and the test material solution were measured at the beginning and at the end of the renewal periods.

RANGE-FINDING STUDY
- A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations could be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions.
- Test concentrations: 0.1, 1, 10 and 100 mg/L solutions preparaed by appropriate dilution of the stock solution.
- Results used to determine the conditions for the definitive study: Yes. Because significant immobility was observed at the highest concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were used in the main experiment under static conditions. The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
18.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % conf. limits: 17.59 – 20.23 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
18.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % conf. limits: 17.59 – 20.23 mg/L
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
53.04 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.:
6.71 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.:
13.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
VALIDITY
- There was no immobilisation in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L.
- All validity criteria were within acceptable limits and therefore the study can be considered as valid.

CONCENTRATIONS OF THE TEST MATERIAL
- Test concentrations were analytically determined at the beginning and at the end of the experiment. Samples from the control were also analysed at the start and at the end of the test.
- The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L.
- The corresponding measured geometric mean test item concentrations were: 6.71, 13.30, 26.75, 53.04 and 104.12 mg/L.
- As the measured concentrations deviated more than 20 per cent from the nominal in some cases, the biological results are related to the geometric mean of the measured test material concentrations.

IMMOBILISATION
- The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour (Table 1).
- In addition to immobility, no abnormal behaviour or appearance of test animals was detected.
Results with reference substance (positive control):
- The 24h EC50 Potassium dichromate: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.68 mg/L)
Reported statistics and error estimates:
- The 24 hours EC50 values of the test material could not be calculated due to the slight effects observed. The 48 hours EC50 values were calculated using Probit analysis by TOXSTAT software. The 24 h EC50 and 48 h EC100 values of the test material were determined directly from the raw data.
- For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

Table 1: Number and percentage of immobilised animals

Concentration (mg/L)

Number of animals

Immobilised animals

24 hours

48 hours

Nominal

Measured

number

percent

number

percent

Control

0.0

20

0

0

0

0

6.25

6.71

20

0

0

0

0

12.5

13.30

20

3

15*

3

15*

25.0

26.75

20

17

85*

17

85*

50.0

53.04

20

20

100*

20

100*

100.0

104.12

20

20

100*

20

100*

* statistically significantly different compared to the control values (Dunnett’s Test; α = 0.05)

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study the 24 and 48-h EC50 was 18.86 mg/L (measured) with 95 % CI of 17.59-20.23 mg/L. The 48-h EC100 was 53.04 mg/L, the 48-h NOEC was 6.71 mg/L and the 48-h LOEC was 13.30 mg/L.
Executive summary:

The toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202, EPA OCSPP 850.1010 and Japanese Guidelines, under GLP conditions.

Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a static test system. Because significant immobility was observed at the examined concentration levels during the preliminary range-finding tests, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the definitive test under static conditions. The test concentrations were analytically determined at the start and at the end of the experiment. The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L. The corresponding measured geometric mean test material concentrations were: 6.71, 13.30, 26.75, 53.04 and 104.12 mg/L. As the measured concentrations deviated more than 20 per cent from the nominal in some cases, biological results are based on the measured geometric mean test material concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentrations and for the control as well.

The 24 and 48 hours EC50 values of the test material were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test material were determined directly from the raw data. For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

All validity criteria were met during this study.

Under the conditions of the study the 24 and 48-h EC50 was 18.86 mg/L (measured) with 95 % CI of 17.59-20.23 mg/L. The 48-h EC100 was 53.04 mg/L, the 48-h NOEC was 6.71 mg/L and the 48-h LOEC was 13.30 mg/L.

 

Description of key information

Under the conditions of the study the 24 and 48-h EC50 was 18.86 mg/L (measured) with 95 % CI of 17.59-20.23 mg/L. The 48-h EC100 was 53.04 mg/L, the 48-h NOEC was 6.71 mg/L and the 48-h LOEC was 13.30 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
18.86 mg/L

Additional information

Sipos, 2018

The toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202, EPA OCSPP 850.1010 and Japanese Guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a static test system. Because significant immobility was observed at the examined concentration levels during the preliminary range-finding tests, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the definitive test under static conditions. The test concentrations were analytically determined at the start and at the end of the experiment. The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L. The corresponding measured geometric mean test material concentrations were: 6.71, 13.30, 26.75, 53.04 and 104.12 mg/L. As the measured concentrations deviated more than 20 per cent from the nominal in some cases, biological results are based on the measured geometric mean test material concentrations.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentrations and for the control as well.

The 24 and 48 hours EC50 values of the test material were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test material were determined directly from the raw data. For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

All validity criteria were met during this study.

Under the conditions of the study the 24 and 48-h EC50 was 18.86 mg/L (measured) with 95 % CI of 17.59-20.23 mg/L. The 48-h EC100 was 53.04 mg/L, the 48-h NOEC was 6.71 mg/L and the 48-h LOEC was 13.30 mg/L.

 

Yoshimura, 2015

The acute toxicity of the test material to Daphnia magna was investigated in accordance with the standardised guideline OECD 202. The study was not performed under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Five daphnids per vessel were treated with different concentrations of test concentration with four duplicates of each. The tested concentrations were: 2.2, 4.8, 10, 23 and 50 mg/L and dilutions were prepared in dilution water.

The loading daphnids were observed to confirm numbers of immobilised daphnids at 24 and 48 hour exposure. Individuals that did were unable to swim within 15 seconds after gentle agitation of the test vessel were considered immobile.

Under the conditions of this study, the 48 hour EC50 for the acute toxicity of the test material to Daphnia magna was 6.60 mg/L with 95 % confidence limits of 4.8 - 10.0 mg/L.