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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:
23 March 2018 to 25 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
The analytical phase of this study was conducted at the Test Site, Seibersdorf Labor GmbH, in Austria.
The concentration of Cadmium, as a marker for the test item was measured at all tested concentration levels at the beginning and at the end of the renewal periods using a suitable ICP method.
Samples were taken from the control and at the applied test concentration levels (three replicates) at the beginning and at the end of the renewal periods.

Duplicate samples were taken in plastic tubes, acidified with pure nitric acid (9 mL sample + 1 mL HNO3) (stored under test conditions until analysis) in agreement with the requirements of the Principal Investigator. To avoid any problems with micro particles, therefore the samples were membrane filtrated before acidification. After sampling, one set of the samples was stored under test conditions and then sent to the Test Site for analysis. One set of the samples is retained as a back-up at the Test Facility, thus these samples were frozen and kept approximately at -20°C at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results
were obtained on the first set of).
Vehicle:
yes
Details on test solutions:
Formulation
Because the test item is very poorly soluble in water, a test solution will be prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity
Testing of Difficult Substances and Mixtures, OECD No. 23.

A saturated test item solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (ISO Medium) two days
before the start of the study. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved
test material was removed by filtration through a fine (0.22 μm) filter to give the 100% saturated solution.
The test solutions were prepared by appropriate dilution of the 100 % saturated solution just before introduction of the Daphnia (start of the treatments).

Due to that stability of the test item was not available the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. Prior to treatment of each renewal period, test item solutions were prepared by the method described above.

Untreated Control
The dilution water (ISO-medium) was used without addition of the test item.

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.

The date of the last study (Study Code: 17/367-023DA) with reference item Potassium dichromate (batch no.: A0345704) is: 06 - 07 December 2017. The 24h EC50: 0.65 mg/L, (95 % confidence limits: 0.61 – 0.70 mg/L)
Test organisms (species):
Daphnia magna
Details on test organisms:
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Breeding The Daphnia are bred in Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: There were 20 animals in test group and control group respectively, divided into 4 replicates (5 animals / replicate)
Age of the animals: They were less than 24 h old at the beginning of the test
Acclimatization: There was no acclimatization because the water used was similar to the culture water.
Test type:
semi-static
Water media type:
other: Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.
Limit test:
yes
Total exposure duration:
48 h
Hardness:
The reconstituted water (ISO medium) had a total hardness of 246 mg/L (as CaCO3).
Test temperature:
The water temperature was measured at the start and at the end of the renewal periods in each test vessel. The test temperature was in the range of 20.1 – 20.6°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 19.8 and 20.7°C.
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 7.15 – 7.78.
Dissolved oxygen:
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 7.1 – 8.6 mg/L.
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
The nominal concentrations of test item used in the main experiment were: 1.0, 3.1, 9.8, 31.3 and 100.0 % saturated solutions.

The corresponding measured geometric mean test item concentrations were: 11.38, 34.36, 107.88, 344.78 and 1146.99 μg/L (based on the calculated Cadmium content of 58.737 % in the test item).
Details on test conditions:
DESCRIPTION OF THE TEST PROCEDURE
The test duration was 48 hours. Twenty animals, divided into four groups (glass beakers) of five animals each (at least 5 mL test solution/animal) were used at the test concentrations and for the control group as well.
The choice of the test concentration was made on the basis of the results of the preliminary range-finding test.

Preliminary Range-Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item 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 (in absence of stability data).

During the formulation procedure the stock solution was prepared by the similar method described above (3.1.2.). The test solutions were prepared by appropriate dilution of the stock solution.
The concentration levels used and results (48 h) of the preliminary range-finding test are summarized in the following tables.

Test item concentrations in the Definitive Test
According to the Study Plan at least five test concentrations in a geometric series using factor not exceeding 2.2 should be used in the main test. However, comparing the preliminary range-finding test with the main test large
differences can be observed at the solubility level of the test item (100 % saturated solution). Because toxic effect was observed at the solubility level of the test item during the first definitive test (which was stopped) and taking into account that the lowest concentration tested should preferably give no effect in the definitive test, therefore a wide concentration range was used; five test concentrations in a geometric series (using factor 3.2) and one control were tested in the definitive test under semi-static conditions.

OBSERVATIONS
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 control and the test solutions were measured at the beginning and at the end of the renewal periods.

STATISTICAL EVALUATION
The 24 and 48 hours EC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test item were determined directly from the raw data.
For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.
Reference substance (positive control):
no
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
241.06 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (95 % conf. limits: 207.18 – 280.49 μg/L)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
31.2 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (95 % conf. limits: 25.71 – 37.87 μg/L)
Key result
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
1 146.99 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
11.38 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
34.36 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
VALIDITY
There was no immobilization 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.

IMMOBILISATION
The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour.
In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Although 5 % immobilization could be observed at the test item group of 1.0 saturated solution, this difference was not statistically significant compared to the control and is negligible due to that the accepted control immobilization is 10 %.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Not specified

Number and percentage of immobilised animals

Concentration

Number

of treated

animals

24 hours

48 hours

Nominal

[% sat. sol.]

Measured

[μg/L]

number

percent

number

percent

Control

0.0

20

0

0

0

0

1.0

11.38

20

0

0

1

5

3.1

34.36

20

0

0

15*

75

9.8

107.88

20

6*

30

18*

90

31.3

344.78

20

10*

50

18*

90

100.0

1146.99

20

20*

100

20*

100

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

Validity criteria fulfilled:
yes
Conclusions:
Acute toxicity of Cadmium selenide was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Cadmium selenide were the followings:
Based on the nominal concentration (% saturated solution of 100 mg/L nominal loading rate):
The 24h EC50 value: 21.76% saturated solution (nominal) (95 % conf. limits: 18.72 – 25.28% saturated solution)
The 48h EC50 value: 2.80% saturated solution (nominal) (95 % conf. limits: 2.30 – 3.40% saturated solution)
The 48h EC100 value: 100.0% saturated solution (nominal)

The 48h No-Observed Effect Concentration (NOEC): 1.0% saturated solution (nominal)
The 48h Lowest Observed Effect Concentration (LOEC): 3.1% saturated solution (nominal)

Based on the measured test item concentrations: The 24h EC50 value: 241.06 μg/L (measured) (95 % conf. limits: 207.18 – 280.49 μg/L)
The 48h EC50 value: 31.20 μg/L (measured) (95 % conf. limits: 25.71 – 37.87 μg/L)
The 48h EC100 value: 1146.99 μg/L (measured)

The 48h No-Observed Effect Concentration (NOEC): 11.38 μg/L (measured)
The 48h Lowest Observed Effect Concentration (LOEC): 34.36 μg/L (measured)
Executive summary:

Acute toxicity of Cadmium selenide on Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system. Even if significant immobility was not observed during the preliminary range-finding test, however, comparing the preliminary range-finding test with the main test (which was stopped) large differences can be observed at the solubility level of the test item (100% saturated solution). Therefore five test concentrations in a geometric series (using factor 3.2) and one control were tested in the definitive test under semi-static conditions.

The nominal concentrations of test item used in the main experiment were: 1.0, 3.1, 9.8, 31.3 and 100.0 % saturated solutions.

The test concentrations were analytically determined at the start and at the end of the renewal periods by measuring the concentration of Cadmium (as a marker for the test item) using a suitable ICP method. The corresponding measured geometric mean test item concentrations were: 11.38, 34.36, 107.88, 344.78 and 1146.99 μg/L (based on the calculated Cadmium content of 58.737 % in the test item).

The biological results are based on both the nominal percentage saturated solutions and measured geometric mean test item 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 item and their confidence limits were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test item were determined directly from the raw data.

For the determination of the LOEC and NOEC, the immobilization 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 this Daphnia magna acute immobilisation study the observed endpoints for the effect of Cadmium selenide were the followings:

Based on the nominal concentration (% saturated solution of 100 mg/L nominal loading rate):

The 24h EC50 value: 21.76% saturated solution (nominal) (95 % conf. limits: 18.72 – 25.28% saturated solution)

The 48h EC50 value: 2.80% saturated solution (nominal) (95 % conf. limits: 2.30 – 3.40% saturated solution)

The 48h EC100 value: 100.0% saturated solution (nominal)

The 48h No-Observed Effect Concentration (NOEC): 1.0% saturated solution (nominal)

The 48h Lowest Observed Effect Concentration (LOEC): 3.1% saturated solution (nominal)

Based on the measured test item concentrations:

The 24h EC50 value: 241.06 μg/L (measured) (95 % conf. limits: 207.18 – 280.49 μg/L)

The 48h EC50 value: 31.20 μg/L (measured) (95 % conf. limits: 25.71 – 37.87 μg/L)

The 48h EC100 value: 1146.99 μg/L (measured)

The 48h No-Observed Effect Concentration (NOEC): 11.38 μg/L (measured)

The 48h Lowest Observed Effect Concentration (LOEC): 34.36 μg/L (measured)

Description of key information

Acute toxicity of Cadmium selenide was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Cadmium selenide were the followings:

Based on the nominal concentration (% saturated solution of 100 mg/L nominal loading rate):

The 24h EC50 value: 21.76% saturated solution (nominal) (95 % conf. limits: 18.72 – 25.28% saturated solution)

The 48h EC50 value: 2.80% saturated solution (nominal) (95 % conf. limits: 2.30 – 3.40% saturated solution)

The 48h EC100 value: 100.0% saturated solution (nominal)

The 48h No-Observed Effect Concentration (NOEC): 1.0% saturated solution (nominal)

The 48h Lowest Observed Effect Concentration (LOEC): 3.1% saturated solution (nominal)

Based on the measured test item concentrations: The 24h EC50 value: 241.06 μg/L (measured) (95 % conf. limits: 207.18 – 280.49 μg/L)

The 48h EC50 value: 31.20 μg/L (measured) (95 % conf. limits: 25.71 – 37.87 μg/L)

The 48h EC100 value: 1146.99 μg/L (measured)

The 48h No-Observed Effect Concentration (NOEC): 11.38 μg/L (measured)

The 48h Lowest Observed Effect Concentration (LOEC): 34.36 μg/L (measured)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
31.2 µg/L

Additional information

Acute toxicity of Cadmium selenide on Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system. Even if significant immobility was not observed during the preliminary range-finding test, however, comparing the preliminary range-finding test with the main test (which was stopped) large differences can be observed at the solubility level of the test item (100% saturated solution). Therefore five test concentrations in a geometric series (using factor 3.2) and one control were tested in the definitive test under semi-static conditions.

The nominal concentrations of test item used in the main experiment were: 1.0, 3.1, 9.8, 31.3 and 100.0 % saturated solutions.

The test concentrations were analytically determined at the start and at the end of the renewal periods by measuring the concentration of Cadmium (as a marker for the test item) using a suitable ICP method. The corresponding measured geometric mean test item concentrations were: 11.38, 34.36, 107.88, 344.78 and 1146.99 μg/L (based on the calculated Cadmium content of 58.737 % in the test item).

The biological results are based on both the nominal percentage saturated solutions and measured geometric mean test item 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 item and their confidence limits were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test item were determined directly from the raw data.

For the determination of the LOEC and NOEC, the immobilization 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 this Daphnia magna acute immobilisation study the observed endpoints for the effect of Cadmium selenide were the followings:

Based on the nominal concentration (% saturated solution of 100 mg/L nominal loading rate):

The 24h EC50 value: 21.76% saturated solution (nominal) (95 % conf. limits: 18.72 – 25.28% saturated solution)

The 48h EC50 value: 2.80% saturated solution (nominal) (95 % conf. limits: 2.30 – 3.40% saturated solution)

The 48h EC100 value: 100.0% saturated solution (nominal)

The 48h No-Observed Effect Concentration (NOEC): 1.0% saturated solution (nominal)

The 48h Lowest Observed Effect Concentration (LOEC): 3.1% saturated solution (nominal)

Based on the measured test item concentrations:

The 24h EC50 value: 241.06 μg/L (measured) (95 % conf. limits: 207.18 – 280.49 μg/L)

The 48h EC50 value: 31.20 μg/L (measured) (95 % conf. limits: 25.71 – 37.87 μg/L)

The 48h EC100 value: 1146.99 μg/L (measured)

The 48h No-Observed Effect Concentration (NOEC): 11.38 μg/L (measured)

The 48h Lowest Observed Effect Concentration (LOEC): 34.36 μg/L (measured)