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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:
19 March 2018 to 21 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex IV Part C, C.3
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, Section 2, No. 202, “Daphnia sp., Acute Immobilisation Test”, updated 13th April 2004.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Version / remarks:
EPA Guideline 712-C-96-114: OCSPP 850.1010, “Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids” April 1996.
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
other: JMAFF 2-7-2-1 The guidelines related to the study reports for the registration application of pesticide
Version / remarks:
JMAFF 2-7-2-1 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:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
other: Japanese National Guidelines
Version / remarks:
Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE).
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
Concentration of Test Item in the test solutions was determined at the beginning and at the end of each renewal period. Control samples were taken only at the beginning of the renewal periods.
Vehicle:
no
Details on test solutions:
Because the Test Item is poorly soluble in water, a test solution was 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 test. This solution was shaken for approximately 24 hours at approximately 30°C and then was equilibrated for approximately 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.
In the lack of stability data for the 48h exposure period the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. Prior to the treatment of the renewal periods the Test Item solutions were prepared by the method described above.
The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (before treatment at each renewal period).
Test organisms (species):
Daphnia magna
Details on test organisms:
Species and strain: Daphnia magna
Source: Szent István University, Department of Aquaculture, 2100 Gödöllő, Páter Károly u. 1. – Hungary
Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: There were 20 animals in test and control groups 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:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not specified
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 each renewal period in each test vessel. The test temperature was in the range of 20.2 – 20.7°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 19.8 – 20.8°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 each renewal period in each test vessel and was in the range of 7.04 – 7.75.
Dissolved oxygen:
The dissolved oxygen concentration was measured in each test vessel at the start and at the end of each renewal period and was in the range of 8.2– 8.7 mg/L.
Salinity:
Not applicable
Conductivity:
Not specified
Nominal and measured concentrations:
The concentrations in the definitive test were based on the results of the preliminary range-finding test and were: 0.54, 1.23, 2.84, 6.52 and 15 % saturated solutions.
Details on test conditions:
PERFORMANCE OF THE TEST
The test duration was 48 hours. Twenty animals, divided into four groups (glass beaker) of five animals each (~8 mL test solution/animal) were used. Taking into account that the 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. The animals were not fed during the test.
The choice of the test concentration was done on the basis of the results of a 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 can 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.
During the formulation procedure the stock solution was prepared. The test solutions were prepared by appropriate dilution of this stock solution.

Test Item concentrations in the Definitive Test
Because significant immobility was observed at the two highest examined concentration levels during the Preliminary Range-Finding Test, five test concentrations in a geometric series (factor 2.3) plus a Control were used in the Main Test under semi-static conditions. The used factor (2.3) exceeded the one recommended in the OECD guideline (2.2) due to the observed toxicity in the Preliminary Range-Finding Test.
The concentrations in the definitive test were based on the results of the preliminary range-finding test and were: 0.54, 1.23, 2.84, 6.52 and 15 % saturated solutions.

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 controls and the test solutions were measured at the beginning and at the end of each renewal period.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.022 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.051 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
VALIDITY
There was no immobilized animal 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 ITEM
Test concentrations were analytically determined at the start and at the end of each renewal periods.
The nominal concentrations of Test Item used in the main experiment were: 0.54, 1.23, 2.84, 6.52 and 15 % saturated solutions. Test concentrations were analytically determined at the start and at the end of each renewal period. The corresponding measured geometric mean Test Item concentrations were: 0.00462, 0.01018, 0.02214, 0.06428, 0.15118 mg/L. There were no Test Item detected in the Control samples.

IMMOBILISATION
The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour.
At the end of the exposure; at the Test Item concentrations of 0.02214 mg/L, 5% immobilisations was observed. This value did not reach statistical significance and are below the control level (10%), therefore were considered to not have a relation to the Test Item.
Results with reference substance (positive control):
The 24h EC50: 0.65 mg/L, (95% confidence limits: 0.61–0.70 mg/L).
Reported statistics and error estimates:
The 24 hours EC50 and 48 hours EC50 values were calculated using Probit analysis by SPSS PC+ software. The 48h EC100 value of the Test Item was determined 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.

Exposure concentrations

Nominal concentrations

(% saturated solution)

Measured concentrations(mg/L)

Measured geometric mean(mg/L)

1strenewal period

2ndrenewal period

Start

End

Start

End

Control

0.54

1.23

2.84

6.52

15

n.d.

0.0133

0.0361

0.0824

0.189

0.442

n.d.

0.00151

0.00223

0.00452

0.02 ¥

0.0310

n.d.

0.0145

0.0412

0.0921

0.213

0.501

n.d.

0.00156

0.00324

0.00700

0.0212

0.0761

--

0.00462

0.01018

0.02214

0.06428

0.15118

n.d.: not detected

¥: this value was <LOQ and during the calculation of measured geometric mean concentration, the LOQ was used which is 0.02 mg/L.

 

Number and percentage ofimmobilisedanimals

Concentration

Number of treated animals

Immobilisedanimals

Nominal

Measured**

24 hours

48 hours

(% saturated solution)

(mg/L)

Number

%

Number

%

Control

0.54

1.23

2.84

6.52

15

n.d.

0.00462

0.01018

0.02214

0.06428

0.15118

20

20

20

20

20

20

0

0

0

1

0

15*

0

0

0

5

0

75

0

0

0

1

13*

20*

0

0

0

5

65

100

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

**: measured geometric mean concentration.

 

DATA OF TEST CONDITIONS

Temperature measured in the test vessels (°C)

Concentration

Replicate

Measuring

Nominal

(% saturated solution)

Measured

(mg/L)

0 h

(fresh media)

24 h

(old media)

24 h

(fresh media)

48 h

(old media)

Control

n.d.

1

2

3

4

20.7

20.7

20.7

20.7

20.2

20.2

20.2

20.2

20.6

20.6

20.6

20.6

20.3

20.3

20.3

20.3

0.54

0.00462

1

2

3

4

20.7

20.7

20.7

20.7

20.2

20.2

20.2

20.2

20.6

20.6

20.6

20.6

20.3

20.3

20.3

20.3

1.23

0.01018

1

2

3

4

20.7

20.7

20.7

20.7

20.2

20.2

20.2

20.2

20.6

20.6

20.6

20.6

20.3

20.3

20.3

20.3

2.84

0.02214

1

2

3

4

20.7

20.7

20.7

20.7

20.2

20.2

20.2

20.2

20.6

20.6

20.6

20.6

20.3

20.3

20.3

20.3

6.52

0.06428

1

2

3

4

20.7

20.7

20.7

20.7

20.2

20.2

20.2

20.2

20.6

20.6

20.6

20.6

20.3

20.3

20.3

20.3

15

0.15118

1

2

3

4

20.7

20.7

20.7

20.7

20.2

20.2

20.2

20.2

20.6

20.6

20.6

20.6

20.3

20.3

20.3

20.3

 

Oxygen concentrations measured in the test vessels (mg/L)

Concentration

Replicate

Measuring

Nominal

(% saturated solution)

Measured

(mg/L)

0 h

(fresh media)

24 h

(old media)

24 h

(fresh media)

48 h

(old media)

Control

n.d.

1

2

3

4

8.6

8.6

8.6

8.6

8.5

8.5

8.4

8.4

8.7

8.7

8.7

8.7

8.4

8.4

8.4

8.4

0.54

0.00462

1

2

3

4

8.5

8.5

8.5

8.5

8.3

8.3

8.3

8.3

8.6

8.6

8.6

8.6

8.4

8.4

8.4

8.5

1.23

0.01018

1

2

3

4

8.5

8.5

8.5

8.5

8.2

8.2

8.3

8.3

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

2.84

0.02214

1

2

3

4

8.5

8.5

8.5

8.5

8.3

8.3

8.3

8.3

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

6.52

0.06428

1

2

3

4

8.5

8.5

8.5

8.5

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.3

15

0.15118

1

2

3

4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.4

 

pH measured in the test vessels

Concentration

Replicate

Measuring

Nominal

(% saturated solution)

Measured

(mg/L)

0 h

(fresh media)

24 h

(old media)

24 h

(fresh media)

48 h

(old media)

Control

n.d.

1

2

3

4

7.12

7.12

7.12

7.12

7.59

7.61

7.62

7.63

7.16

7.16

7.16

7.16

7.04

7.08

7.10

7.11

0.54

0.00462

1

2

3

4

7.13

7.13

7.13

7.13

7.63

7.63

7.63

7.63

7.38

7.38

7.38

7.38

7.11

7.12

7.12

7.12

1.23

0.01018

1

2

3

4

7.15

7.15

7.15

7.15

7.65

7.65

7.66

7.66

7.40

7.40

7.40

7.40

7.13

7.13

7.13

7.14

2.84

0.02214

1

2

3

4

7.18

7.18

7.18

7.18

7.67

7.67

7.67

7.67

7.39

7.39

7.39

7.39

7.15

7.16

7.16

7.17

6.52

0.06428

1

2

3

4

7.20

7.20

7.20

7.20

7.69

7.69

7.69

7.71

7.39

7.39

7.39

7.39

7.17

7.17

7.19

7.19

15

0.15118

1

2

3

4

7.23

7.23

7.23

7.23

7.73

7.73

7.75

7.75

7.41

7.41

7.41

7.41

7.20

7.20

7.20

7.21

 

DATA OF IMMOBILISATION

Immobilisationof the test animals

Concentration

Replicate

Number of treated animals

Number of immobilized animals

Nominal

(% saturated solution)

Measured

(mg/L)

24 h

48 h

Control

n.d.

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0.54

0.00462

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

1.23

0.01018

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

2.84

0.02214

1

2

3

4

5

5

5

5

1

0

0

0

1

0

0

0

6.52

0.06428

1

2

3

4

5

5

5

5

0

0

0

0

3

4

3

3

15

0.15118

1

2

3

4

5

5

5

5

4

4

3

4

5

5

5

5

 

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the study, the calculated endpoints for the effect of the Test Item were the following:

EC50 48h: 0.0512 mg/L
NOEC 48h: 0.00214 mg/L
Executive summary:

Acute toxicity of Test Item was assessed with Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

 

Based on the performed preliminary range-finding test five test concentrations in a geometric series with a separation factor of 2.3 and one untreated Control were tested in the main experiment.

 

The nominal concentrations of Test Item used in the main experiment were: 0.54, 1.23, 2.84, 6.52 and 15 % saturated solutions. Test concentrations were analytically determined at the start and at the end of each renewal period. The corresponding measured geometric mean Test Item concentrations were: 0.00462, 0.01018, 0.02214, 0.06428, 0.15118 mg/L. The biological results are based on the measured and calculated concentrations.

 

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

 

The 24 hours EC50 and 48 hours EC50 values were calculated using Probit analysis by SPSS PC+ software. The 48h EC100 value of the Test Item was determined 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.

 

In conclusion, under the conditions of this study, the calculated endpoints for the effect of the Test Item were the following:

Summary of endpoints

Parameter

(48 hours)

EC50

(measured)

EC100

(measured)

NOEC

(measured)

LOEC

(measured)

 

0.0512 mg/L

0.15118 mg/L

0.02214 mg/L

0.06428 mg/L

95% conf. limits

0.0396 – 0.0648 mg/L

--

--

--

 

Description of key information

The calculated endpoints for the effect of the Test Item were the following:

Summary of endpoints

Parameter

(48 hours)

EC50

(measured)

EC100

(measured)

NOEC

(measured)

LOEC

(measured)

 

0.0512 mg/L

0.15118 mg/L

0.02214 mg/L

0.06428 mg/L

95% conf. limits

0.0396 – 0.0648 mg/L

--

--

--

 

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.051 mg/L

Additional information

Acute toxicity of Test Item was assessed with Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

 

Based on the performed preliminary range-finding test five test concentrations in a geometric series with a separation factor of 2.3 and one untreated Control were tested in the main experiment.

 

The nominal concentrations of Test Item used in the main experiment were: 0.54, 1.23, 2.84, 6.52 and 15 % saturated solutions. Test concentrations were analytically determined at the start and at the end of each renewal period. The corresponding measured geometric mean Test Item concentrations were: 0.00462, 0.01018, 0.02214, 0.06428, 0.15118 mg/L. The biological results are based on the measured and calculated concentrations.

 

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

 

The 24 hours EC50 and 48 hours EC50 values were calculated using Probit analysis by SPSS PC+ software. The 48h EC100 value of the Test Item was determined 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.

 

In conclusion, under the conditions of this study, the calculated endpoints for the effect of the Test Item were the following:

Summary of endpoints

Parameter

(48 hours)

EC50

(measured)

EC100

(measured)

NOEC

(measured)

LOEC

(measured)

 

0.0512 mg/L

0.15118 mg/L

0.02214 mg/L

0.06428 mg/L

95% conf. limits

0.0396 – 0.0648 mg/L

--

--

--