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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Threshold Concentration approach adopted - single limit concentration applied. No mortality nor sub-lethal effects observed - i.e. 96 hour LC50 likely to be significantly higher than 12 mg/L.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
Threshold concentration test
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
Threshold concentration test
Principles of method if other than guideline:
The OECD 201 and OECD 202 studies were performed prior to this study. The lowest EC50 value derived from these studies (in this case the EyC50 from the OECD 201 Algal Inhibition study) was employed to fix a single threshold concentration to cinduct the study, in the interests of animal wellfare and according to the OECD Guidance Document : Short guidance on the threshold approach for acute fish toxicity testing. Series on testing and assessment No. 126 (2010).
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
FRESKOMENTHE
Lot No. VE00426966
Purity 99.2%
Expiry Date 19 February 2018
Analytical monitoring:
yes
Remarks:
GC-FID
Details on sampling:
The limit concentration and the control were analytically verified via GC-FID from freshly prepared media after 0 and 72 h and from corresponding 24 h aged test media after 24 and 96 h.
Vehicle:
no
Details on test solutions:
12.0 mg/L was tested as a threshold concentration in a limit test. Taking into account the aquatic toxicity data of the test item the test was performed without a range-finding test. The threshold concentration (TC) was based on the toxicity data obtained by an acute daphnia toxicity test (EC50, 48h = 25 mg/L, nominal concentration) and alga toxicity test (ErC50 (0-72 h) = 30.6 mg/L, EyC50 (0-72 h) = 12.2 mg/L, nominal concentration) both KLIX, 2017.
An appropriate volume of the test item was pipetted directly into a test vessel filled with an appropriate volume of dilution water. The test media were mixed with an ultraturrax (1 min, 12000 rpm). This procedure was repeated for every water renewal.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Holding was performed at the test facility at 23 ± 2°C and diffuse light (7 – 750 Lux, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days prior to the start of exposure were used for testing. No disease treatments were administered throughout holding and testing.
Test type:
semi-static
Water media type:
freshwater
Remarks:
Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Limit test:
yes
Total exposure duration:
96 h
Hardness:
67 mg/L as CaCO3
Test temperature:
Min. 22.1 °C;
Max. 23.0 °C;
Mean temperature +/- Standard Deviation : 22.5 +/- 0.2 °C
pH:
pH 7.32 - 7.73
Dissolved oxygen:
78 - 100% Saturation
Nominal and measured concentrations:
Nominal Concentration of Test Item = 12.0 mg/L
Geometric Mean Measured Concentration = 10.7 mg/L
Details on test conditions:
A semi-static test with daily renewal of the test media was performed. With regard to the properties of the test item, the test aquaria were covered with a floating lid (i.e. with aluminum foil) to reduce the loss of the test item via the surface.
12.0 mg/L was tested as a threshold concentration in a limit test. Taking into account the aquatic toxicity data of the test item the test was performed without a range-finding test. The threshold concentration (TC) was based on the toxicity data obtained by an acute daphnia toxicity test (EC50, 48h = 25 mg/L, nominal concentration) and alga toxicity test (ErC50 (0-72 h) = 30.6 mg/L, EyC50 (0-72 h) = 12.2 mg/L, nominal concentration) both KLIX, 2017.
Control - 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicate.
Test Vessel - Glass aquaria of 13 L were used, filled with approximately 10 L of dilution water (dimensions: 25.5/18/28 cm, depth of water: 20 cm), covered with a floating lid (e.g. with aluminum foil), to reduce the loss of the test item, containing 10 L of water.
Replication - One replicate for the limit concentration and the control.
The fish density in the tanks was 0.19 g fish per litre test solution. Average body length at the test end: 2.74 cm (2.5 – 3.0 cm). Average body weight at the test end: 0.277 g (measurement of all fish together). The measurements of size and weight were carried out at the end of exposure from the control fish.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
>= 10.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
No mortalities observed over the course of the 96 hours exposure at threshold test concentration
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
>= 12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
No mortalities observed over the course of the 96 hours exposure at threshold test concentration
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 10.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
Sub-lethal physical and behavioural observations
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other:
Remarks:
Sub-lethal physical and behavioural observations
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No mortalities observed over the course of the 96 hours exposure at threshold concentration
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 10.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No mortalities observed over the course of the 96 hours exposure at threshold concentration
Details on results:
The LC0 and NOEC corresponds to the nominal Threshold Concentration ≥ 12.0 mg/L and the geometric mean measured Threshold Concentration ≥ 10.7 mg test item/L No mortalities or sub-lethal effects were observed over the course of the 96 hours exposure at threshold concentration : 96 hour LC50 > 12.0 mg/L (nominal) / 10.7 mg/L (measured).

Validity Criteria :
• The O2 saturation was  60 % (measured range: 78 – 100 %).
• The mortality in the control was 0 %.
• The pH-value in the control group did not deviate by more than one unit over the test period (measured range: 7.32 – 7.73).
Results with reference substance (positive control):
Not applicable - no Reference Toxicity standard used.
Validity criteria fulfilled:
yes
Conclusions:
A semi-static test was performed as a threshold concentration test with a concentration 12 mg/L, corresponding to the geometric mean measured test item concentration of 10.7 mg/L. The threshold concentration of 12 mg/L (nominal) was chosen based on the lowest EC50 value derived from the OECD 201 and OECD 202 studies on FRESKOMENTHE and corresponds to the 72 hour EC50 determined for yield inhibition of algae.
No mortality nor sub lethal effects were observed for fish exposed to either the control nor the threshold concentration throughout the 96 hour duration of the study. The LC100 and LC50 are > 12.0 mg/L (nominal) and the LC0 and NOEC are both ≥ 12.0 mg/L (nominal).
All effect levels are given based on the nominal and geometric mean measured limit concentration of the test item FRESKOMENTHE. The limit concentration of the test item was found to have no lethal and sub lethal effects on zebrafish.
In conclusion, the LC0 and NOEC corresponds to the nominal Threshold Concentration ≥ 12.0 mg/L and the geometric mean measured Threshold Concentration ≥ 10.7 mg test item/L.
Executive summary:

A semi-static test was performed as a threshold concentration test with a concentration 12 mg/L, corresponding to the geometric mean measured test item concentration of 10.7 mg/L. The threshold concentration of 12 mg/L (nominal) was chosen based on the lowest EC50 value derived from the OECD 201 and OECD 202 studies on FRESKOMENTHE and corresponds to the 72 hour EC50 determined for yield inhibition of algae.

No mortality nor sub lethal effects were observed for fish exposed to either the control nor the threshold concentration throughout the 96 hour duration of the study. The LC100 and LC50 are > 12.0 mg/L (nominal) and the LC0 and NOEC are both ≥ 12.0 mg/L (nominal).

All effect levels are given based on the nominal and geometric mean measured limit concentration of the test item FRESKOMENTHE. The limit concentration of the test item was found to have no lethal and sub lethal effects on zebrafish.

In conclusion, the LC0 and NOEC corresponds to the nominal Threshold Concentration ≥ 12.0 mg/L and the geometric mean measured Threshold Concentration ≥ 10.7 mg test item/L.

Description of key information

A semi-static test was performed as a threshold concentration test with a concentration 12 mg/L, corresponding to the geometric mean measured test item concentration of 10.7 mg/L. The threshold concentration of 12 mg/L (nominal) was chosen based on the lowest EC50 value derived from the OECD 201 and OECD 202 studies on FRESKOMENTHE and corresponds to the 72 hour EC50 determined for yield inhibition of algae.

No mortality nor sub lethal effects were observed for fish exposed to either the control nor the threshold concentration throughout the 96 hour duration of the study. The LC100 and LC50 are > 12.0 mg/L (nominal) and the LC0 and NOEC are both ≥ 12.0 mg/L (nominal).

All effect levels are given based on the nominal and geometric mean measured limit concentration of the test item FRESKOMENTHE. The limit concentration of the test item was found to have no lethal and sub lethal effects on zebrafish.

In conclusion, the LC0 and NOEC corresponds to the nominal Threshold Concentration ≥ 12.0 mg/L and the geometric mean measured Threshold Concentration ≥ 10.7 mg test item/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
12 mg/L

Additional information