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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:
Experimental start date 06 September 2016 Experimental completion date 13 October 2016
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 (incl. QA statement)
Specific details on test material used for the study:
Identification: Cyclohexanone, peroxide (CAS 12262-58-7)
Physical state/Appearance: Clear colorless liquid
Batch: 1405426797
Purity: 33.9%
Expiry Date: 29 September 2017
Storage Conditions: Approximately 4 °C in darkness
Analytical monitoring:
yes
Details on sampling:
Range-finding test: nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L. A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions.
Defnitive test: 6.25, 12.5, 25, 50 and 100 mg/L. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours

The test samples were thawed with the aid of a water bath then a volume (5 mL), with the addition of water (5 mL) was pipetted into a 20 mL volumetric flask and diluted to volume with acetonitrile.

The samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
Range-finding test
A nominal amount of test item (100 mg) was dissolved in 1 liter of test water, with the aid of prolonged, slow stirring for 24 hours via magnetic stirrer, to give the 100 mg/L test concentration. A series of dilutions was made from the 100 mg/L test concentration to give further test concentrations of 10, 1.0 and 0.10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive test
A nominal amount of test item (200 mg) was dissolved in 2 liters of test water, with the aid of prolonged, slow stirring for 24 hours via magnetic stirrer, to give the 100 mg/L test concentration. A series of dilutions was made from the 100 mg/L test concentration to give further test concentrations of 50, 25, 12.5 and 6.25 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Annex 2) in a temperature controlled room maintaining the water temperature at 18 °C to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
Test temperature:
Recorded daily
pH:
Recorded at start and termination of the test:
7.8 -7.9 at start, 8-.0 - 8.1 at termination
Dissolved oxygen:
Recorded at start and termination of the test.
8.6 - 8.7 mg O2/L at start, 8.8 -9.0 mg O2/L at termination
Salinity:
Not applicable
Nominal and measured concentrations:
Range-finding test: 0.1, 1.0, 10 and 100 mg/l (nominal test concentrations)

Definitive test: 6.25, 12.5, 25, 50 and 100 mg/L (nominal test concentrations)
Details on test conditions:
Range finding test:
10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 °C to 22 °C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids was recorded.
The control group was maintained under identical conditions, but not exposed to the test item.


Definitive test
As in the range-finding test, 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test, 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 °C to 22 °C with a maximum deviation of ± 1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions, but not exposed to the test item.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
18 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
25 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Range-finding Test

No immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L, however, immobilization was observed at 100 mg/L.

Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 94% to 97% of nominal concentrations. There was no significant change in the measured concentrations at 48 hours indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 95% to 99% of nominal concentrations, and so the results are based on nominal test concentrations.

Immobilization Data
Analysis of the immobilization data by geometric mean method at 24 and 48 hours based on the nominal test concentrations gave the following results:
Time (h)
EC50 (mg/L) 95% Confidence limits
(mg/L)
24 18 12.5 - 25
48 18 12.5 - 25
The No Observed Effect Concentration after 24 and 48 hours exposure was 12.5 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 25 mg/L.

Sub-Lethal Effects
No sub-lethal effects of exposure were observed in all test groups.
Results with reference substance (positive control):
Please refer to "Any other information of results" below:

A positive control (Envigo Study Number XT01HS) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All analysis was carried out using the ToxRat Professional computer software package (ToxRat).

Time Point

(Hours)

EC50
(mg/L)

95% Confidence Limits (mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

24

1.3

1.0

-

1.8

1.0

1.8

48

1.2

1.1

-

1.3

0.56

1.0

 

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Validation Criteria

The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress, and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria

Temperature was maintained at 21°C to 22°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Throughout the test the light intensity was observed to be in the range 318 to 446 Lux.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Table1            Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal
Concentration
(mg/L)

Observations (Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

Control

0

10 N

0

10 N

0.10

0

10 N

0

10 N

1.0

0

10 N

0

10 N

10

0

10 N

0

10 N

100

10

A/I

10

A/I

Table2            Cumulative Immobilization Data and Observations in the Definitive Test

Nominal
Concentration
(mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

6.25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

12.5

0

0

0

0

0

0

5 N

5 N

5 N

5 N

25

5

5

5

5

20

100

A/I

A/I

A/I

A/I

50

5

5

5

5

20

100

A/I

A/I

A/I

A/I

100

5

5

5

5

20

100

A/I

A/I

A/I

A/I

 

Nominal
Concentration
(mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

6.25

0

0

0

0

0

0

5 N

5 N

5 N

5 N

12.5

0

0

0

0

0

0

5 N

5 N

5 N

5 N

25

5

5

5

5

20

100

A/I

A/I

A/I

A/I

50

5

5

5

5

20

100

A/I

A/I

A/I

A/I

100

5

5

5

5

20

100

A/I

A/I

A/I

A/I


N = No sub-lethal effects observed

A/I = All daphnia immobilized

Validity criteria fulfilled:
yes
Executive summary:

 Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No. 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours at a temperature of 21°C to 22°C under static test conditions. The number of immobilized daphnia was recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 95% to 99% of nominal concentration and so the results are based on nominal test concentrations.

Exposure of Daphnia magna to the test item gave the following results:

Time Point

(Hours)

EC50
(mg/L)

95% Confidence Limits (mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

48

18

12.5

-

25

12.5

25

 

Description of key information

An OECD 202 study is available with a 48h-EC50 for Daphnia magna of 18.0 mg/L (measured concentration).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
18 mg/L

Additional information

One study performed under GLP according to OECD guideline 202 is available. This resulted in a 48h-EC50 of 18.0 mg/L (measured concentration) for Daphnia magna.

The study is valid without restriction and can therefore be used for C&L and Hazard- and Risk Assessment.