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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:
From February 21st to February 24th 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to the OECD Guideline for Testing of Chemicals No. 202, "Daphnia sp., Acute Immobilisation Test", referenced as Method C.2 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266. In addition, the procedures were designed to comply with the methods of the following guidelines: - ISO 6341. (2012). Water quality-Determination of the inhibition of the mobility of Daphnia magna Strauss (Cladocera, Crustacea)-Acute toxicity test. Third edition, ISO, Geneve, Switzerland; - OECD Series on testing and assessment, No. 23, “Guidance document on aquatic toxicity testing of difficult substances and mixtures”, December 15, 2000.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Inspection by COFRAC on July 4th, 5th and 6th 2016, signed on January 10th 2017
Specific details on test material used for the study:
No additional information
Analytical monitoring:
yes
Details on sampling:
Chemical analyses : Single samples for analysis were taken from the control and all test concentrations at the start of the test (t=0h) and at the end of the test (t=48h).
- Nominal concentrations : 1.0, 2.2, 4.8, 10.6 and 23.5 mg test item/L
- Sampling method: The test samples were injected directly after a dilution by a factor of two with acetone (500µL sample + 500 µL of cyclohexanone 10 mg/L in acetone).
- Sample storage conditions before analysis: The samples were injected directly after their preparation to avoid any degradation of the test item.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the results of a range-finding test, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water to obtain the following nominal concentrations : 1.0, 2.2, 4.8, 10.6 and 23.5 mg test item/L
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): The solvent used for the preparation of the spiking solutions was acetone.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Stock solutions of the test substance were prepared in acetone at concentrations of approximately 1 g/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Age at test start: < 24 hours old
- Female
- No feeding during test
- Food type: suspension of algal cells of Pseudokirchneriella subcapitata
- Amount: up to 0.1-0.2 mg C.Daphnia.-1day.-1.
- Frequency: Daphnids were fed at least three times a week during the breeding period
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
Approximately 250 mg/L as CaCO3
Test temperature:
20 °C +/- 2 °C
pH:
7.79 - 8.55
Dissolved oxygen:
8.04 - 8.95 mg/L
Salinity:
Not applicable
Conductivity:
Not mentionned
Nominal and measured concentrations:
Nominal concentrations : Test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water to obtain the following nominal concentrations (spaced by a factor of approximately 2.2) in agreement with the Sponsor and the Study Monitor: 1.0, 2.2, 4.8, 10.6 and 23.5 mg test item.L-1 [= the highest test concentration was expected to be close to the solubility of the test item in test water; after two weeks of slow-stirring, the concentration of the stock solution of the test item was 16.6 mg.L-1 (mean of 3 replicates), and since concentrations values were calculated considering the test item major constituents included in the main chromatographic peak (representing 70.76% of the test item): 16.6/70.76% = 23.5 mg.L-1].

Measured concentrations : measured concentrations were slightly higher compared to their expected nominal values (up to 126% of expected nominal concentration at 4.8 mg./L). Since the test item was a UVCB substance, the results were based on the nominal test concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass test tubes of 20 mL capacity sealed with screw caps
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: All-glass test tubes of 20 mL capacity sealed with screw caps, each vessel was completely filled with test solution and without headspace
- Aeration: No aeration of the test solutions occurred throughout the test
- No. of organisms per vessel: 5 daphnids per vessel
- No. of vessels per concentration (replicates): 4 replicates with daphnids per treatment group (including controls)
- Biomass loading rate: Not mentionned

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202. The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg/L (as CaCO3).
- Total organic carbon: Control solutions: TOC: 1 and 40 mg Carbon/L

- Culture medium different from test medium: The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Test water used : reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202
- Intervals of water quality measurement: Not mentionned

OTHER TEST CONDITIONS
- Adjustment of pH: The test was carried out without adjustment of the pH.
- Photoperiod: 16h light : 8h dark
- Light intensity: not mentionned

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 0.32, 1.00 and 3.20 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7) was used as reference substance
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
5.163 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL : 6.405 – 8.406 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
7.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL : not determined due to mathematical reasons or inappropriate data
Details on results:
See tables of results included in "Any other information on results incl. tables".

Analytical results:
The analytical results showed that concentrations of test solutions were overall stable. Indeed, specific analyses of samples revealed that test item levels found were satisfactorily maintained within or close to ± 20% of the initial concentration between the start and the end of the test. TOC analysis confirmed that test concentrations were well maintained throughout the test. However, measured concentrations were slightly higher compared to their expected nominal values (up to 126% of expected nominal concentration at 4.8 mg/L). Since the test item was a UVCB substance, the results were based on the nominal test concentrations.After 48 hours of exposure, immobilisations were 0% at 1.0 and 2.2mg/L, 30% at 4.8 mg/L and 100% at 10.6 and 23.5mg test item/L.

Biological results :
In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.
After 24 hours of exposure, immobilisations were 0% at 1.0, 2.2 and 4.8 mg/L, 95% at 10.6 mg/L and 100% at 23.5 mg test item/L.
After 48 hours of exposure, immobilisations were 0% at 1.0 and 2.2 mg/L, 30% at 4.8 mg/L and 100% at 10.6 and 23.5 mg test item/L.
Therefore, the highest concentration without effect after 48 hours was 1.90 mg/L and the lowest concentration with 100% immobilisation was 8.90 mg/L.
The 24 and 48-hour EL50 were determined by the computer program ToxRat using Probit-analysis.
Results with reference substance (positive control):
- ECx: The 24h-EC50 was 1.22 mg/L. The sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg/L to 2.1 mg/L)

Table 1 : Dissolved oxygen concentrations (mg.L-1) during the final test. 

 

Nominal concentration(mg test item.L-1)

Control

1.0

2.2

4.8

10.6

23.5

Start t=0h

8.32

8.24

8.28

8.38

8.28

8.22

End t=48h

8.95

8.14

8.04

8.38

8.47

8.25

All these dissolved-oxygen concentrations correspond to values > 90% of the air-saturation value.

Table 2: Concentrations of the test substance in test water - Final test (main chromatographic peak, representing 70.76% of the test item)

Nominal

concentration

(mg test item.L-1)

Expected

nominal

concentration

of the main chromatographic peak

(mg.L-1)

Start

(t=0h)

End

(t=48h)

Relative loss to initial value (t=0h - t=48h) (%)

Geometric mean measured concentration

Meas. Conc (mg.L-1)

%

Meas. Conc (mg.L-1)

%

mg.L-1

%

Control

0

Abs.

N.A.

Abs.

N.A.

 N.A.

N.A.

N.A.

1.0

0.71

 0.95*

134

 0.79*

111

17

0.87

123

2.2

1.56

2.23

143

 1.62*

104

27

1.90

122

4.8

3.40

4.67

137

3.99

117

15

4.30

126

10.6

7.50

10.08

134

7.79

104

23

8.90

119

23.5

16.6

16.06

97

13.27

80

17

14.60

88

*Values below the LOQ but above the LOD.

N.A.: not applicable

Absence: concentrations below the LOQ (2.08 mg.L-1) and the LOD (0.62 mg.L-1).

Presence: concentrations below the LOQ (2.08 mg.L-1) and above the LOD (0.62 mg.L-).

% = Percent of expected nominal concentration of the main chromatographic peak

Table 3 : Acute immobilisation of daphnids after 24 and 48 hours in the final test: 

Nominal Concentration(mg test item.L-1)

Geometric mean measured concentration

(mg test item.L-1)

Replicate

Number of daphnids exposed

Response at 24h

Response at 48h

Number

Total %

Number

Total %

Control

Control

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

1.0

0.87

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

2.2

1.90

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

4.8

4.30

1

2

3

4

5

5

5

5

0

0

0

0

0

1

1

2

2

30

10.6

8.90

1

2

3

4

5

5

5

5

4

5

5

5

95

5

5

5

5

100

23.5

14.60

1

2

3

4

5

5

5

5

5

5

5

5

100

5

5

5

5

100

Validity criteria fulfilled:
yes
Remarks:
In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress. Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels
Conclusions:
The toxic effect of test item 1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidised to the freshwater invertebrate Daphnia magna was investigated in a closed static test. Under the experimental conditions and based on the nominal test concentrations, the 48-hour EC50 value estimated was 5.163 mg/L.
Executive summary:

This study was performed to assess the acute toxicity of test item 1,5,10 -trimethylcyclododeca-1,5,9 -triene, epoxidised to the freshwater invertebrate Daphnia magna.

The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphniasp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended by Commission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

The criterion measured was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solutionof the test item over a range of nominaltest concentrations of 1.0, 2.2, 4.8, 10.6 and 23.5mg.L-1(a concentration close to the solubility of the test item in test water).

The immobility of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test item were determined by (specific and non-specific) chemical analyses at the start (t=0h) and the end of the test (t=48h).

The analytical results showed that concentrations of test solutions were overall stable. Indeed, specific analyses of samples revealed that test item levels found were satisfactorily maintained within or close to ± 20% of the initial concentration between the start and the end of the test. TOC analysis confirmed that test concentrations were well maintained throughout the test. However, measured concentrations were slightly higher compared to their expected nominal values (up to 126% of expected nominal concentration at 4.8 mg.L-1). Since the test item was a UVCB substance, the results were based on the nominal test concentrations. After 48 hours of exposure, immobilisations were 0% at 1.0 and 2.2mg.L-1, 30% at 4.8 mg.L-1 and 100% at 10.6 and 23.5 mg test item.L-1.

Under the experimental conditions and based on the nominal test concentrations, the 48-hour EC50 value estimated was 5.163 mg/L.

Description of key information

EU method C.2, OECD guideline 202, key study, GLP, validity 1 :

48h-EC50 (Daphnia magna) = 5.163 mg/L based on nominal concentrations (as UVCB substance)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
5.163 mg/L

Additional information

One key study is available to assess the 48h acute toxicity of the registered substance to Daphnia magna, under static conditions, according to the EU Method C.2 and the OECD Guideline 202 with GLP statement. Twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item over a range of nominal test concentrations of 1.0, 2.2, 4.8, 10.6 and 23.5 mg/L. Additionally, a control group was tested in parallel. Since the test item was a UVCB substance, the results were based on these nominal test concentrations. All-glass test tubes of approximately 20 mL capacity were used, sealed with screw caps.

The concentrations of the test item were determined by (specific and non-specific) chemical analyses at the start (t=0h) and the end of the test (t=48h). The analytical results showed that concentrations of test solutions were overall stable.

Validity criteria were fulfilled : In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress ; dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels.

The immobility of the daphnids was determined in a closed static 48-hour test by visual observation after 24 and 48 hours. The assessment of the biological effects was based on the average of measured exposure concentrations (geometric means). After 48 hours of exposure, immobilisations were 0% at 1.0 and 2.2 mg/L, 30% at 4.8 mg/L and 100% at 10.6 and 23.5 mg test item/L.

Under the experimental conditions and based on the nominal test concentrations, the 48-hour EC50 value estimated was 5.163 mg/L