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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:
22 March 2017 to 24 March 2017
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)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002
Version / remarks:
The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE). 2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF 2-7-2-1
Version / remarks:
The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- The test concentrations were analytically determined at the start and at the end of the first and at the last renewal periods.
- Analytical measurements were performed from the control and at the applied test concentration levels at the beginning and at the end of the renewal periods.
- The samples were analysed by a LC-MS method.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Even if the test item seems to be soluble in ISO medium, but strong precipitation is observed within minutes, it is analytically confirmed that test item does not present in
the solution anymore. For this reason the experiment was carried out in a different media, in which we could keep the test item in solution.
- Due to problems with preparation of test material stock solution (strong precipitation) 1:1 v/v solution of ISO medium and deionised water was used as a test medium instead of ISO medium.
- A saturated test material solution (nominal loading rate of 100 mg/L) was prepared with direct addition of the test material, mixed into the test medium (1:1 v/v solution of ISO medium and deionised water) using ultrasonic bath (10 minutes). Saturated test material solution was opalescent. The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of the treatments).
- Prior to the treatment, at each of the renewal periods test solutions were prepared by the method described above.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- 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:
semi-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:
20.6 to 21.0 °C
pH:
6.93 to 7.59.
Dissolved oxygen:
8.0 to 9.1 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solutions.
- Measured geometric mean test material concentrations were: 3.56, 5.48, 11.19, 22.64 and 39.60 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers
- Renewal rate of test solution: The frequency of the water renewal periods was 24 hours due to precipitation observed after 24 hours during the preliminary range-finding test.
- 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 (1:1 v/v solution of ISO medium and deionised water) was used as dilution water for both the range finding and definitive tests.
- Reconstituted water (ISO medium) 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 6.93 – 7.63.
- Photoperiod: The test was carried out in 16-hour light and 8-hour dark cycle.
- Temperature: The water temperature was measured at the start and at the end of the renewal in each test vessel. The test temperature was in
the range of 20.6 – 21.0°C measured in the test vessels. The additionally measured temperature in the climate chamber was between 20.5 and 21.2°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 8.0 – 9.1 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.

VEHICLE CONTROL PERFORMED: Yes

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 % saturated solutions preparaed by appropriate dilution of the stock solutions.
- Results used to determine the conditions for the definitive study: Yes. At 100 % saturated solution, all of the daphnia were immobilised in the preliminary test.
- Due to precipitation observed after 24 hours during the preliminary range-finding test the experiment was performed under semi-static conditions.
- Because significant immobility was observed during the preliminary concentration range-finding test, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the main experiment under semi-static conditions.
- The biological results are based on the measured geometric mean test material concentrations
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 39.6 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.:
26.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (95 % CI: 22.41 to 31.57 mg/L)
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 39.6 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.:
22.64 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.:
39.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
CONCENTRATIONS OF THE TEST MATERIAL
- Test concentrations were analytically determined at the beginning and at the end of the renewal periods. Samples from the control were analysed at the start and at the end of the renewal periods as well.
- The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.
- The corresponding measured geometric mean test item concentrations were: 3.56, 5.48, 11.19, 22.64 and 39.60 mg/L.
- The biological results are based on the measured geometric mean 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.
- Although 10 % immobilisation could be observed at the test item groups of 6.25, 12.5, 25.0 and 50.0 % saturated solution, these differences were not statistically
significant compared to the control and are negligible due to that the accepted control immobilisation is 10 %.
Results with reference substance (positive 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: 17/058-023DA) with reference material Potassium dichromate (batch no.: A0345704) is: 08 - 09 March 2017.
- The 24h EC50: 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

3.56

20

2

10

2

10

12.5

5.48

20

0

0

2

10

25.0

11.19

20

1

5

2

10

50.0

22.64

20

0

0

2

10

100.0

39.60

20

2

10

19

95*

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

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.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study the 48-h EC50 was 26.60 mg/L (measured) with 95 % CI of 22.41 to 31.57 mg/L. The 48-h EC100 was > 39.60 mg/L, the 48-h NOEC was 22.64 mg/L and the 48-h LOEC was 39.60 mg/L.
Executive summary:

The acute toxicity of the test material was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2, EPA OCSPP 850.1010 and Japanese guidelines under GLP conditions. Testing was performed on Daphnia magna was in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system.

Even though the test material seems to be soluble in the test media, strong precipitation was observed within minutes. For this reason the experiment was carried out in a different media. A 1:1 v/v solution of ISO medium and deionised water was used as a test medium instead of ISO medium.

Because significant immobility was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the definitive test under semi-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 % saturated solution.

The test concentrations were analytically determined at the start and at the end of the renewal periods. The corresponding measured geometric mean test material concentrations were: 3.56; 5.48; 11.19; 22.64 and 39.60 mg/L. Biological results are based on the measured geometric mean test material concentrations.

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

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.

All validity criteria were met during this study.

Under the conditions of the study the 48-h EC50 was 26.60 mg/L with 95 % CI of 22.41 to 31.57 mg/L. The 48-h EC100 was > 39.60 mg/L, the 48-h NOEC was 22.64 mg/L and the 48-h LOEC was 39.60 mg/L.

Description of key information

Under the conditions of the study the 48-h EC50 was 26.60 mg/L with 95 % CI of 22.41 to 31.57 mg/L. The 48-h EC100 was > 39.60 mg/L, the 48-h NOEC was 22.64 mg/L and the 48-h LOEC was 39.60 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
26.6 mg/L

Additional information

The acute toxicity of the test material was investigated in accordance with the standardised guidelines OECD 202, EU Method C.2, EPA OCSPP 850.1010 and Japanese guidelines under GLP conditions. Testing was performed on Daphnia magna was in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Even though the test material seems to be soluble in the test media, strong precipitation was observed within minutes. For this reason the experiment was carried out in a different media. A 1:1 v/v solution of ISO medium and deionised water was used as a test medium instead of ISO medium.

Because significant immobility was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the definitive test under semi-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 % saturated solution. The test concentrations were analytically determined at the start and at the end of the renewal periods. The corresponding measured geometric mean test material concentrations were: 3.56; 5.48; 11.19; 22.64 and 39.60 mg/L. Biological results are based on the measured geometric mean test material concentrations.

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

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.

All validity criteria were met during this study.

Under the conditions of the study the 48-h EC50 was 26.60 mg/L with 95 % CI of 22.41 to 31.57 mg/L. The 48-h EC100 was > 39.60 mg/L, the 48-h NOEC was 22.64 mg/L and the 48-h LOEC was 39.60 mg/L.