Registration Dossier
Registration Dossier
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 941-809-7 | CAS number: -
Short-term toxicity to fish
Administrative data
Link to relevant study record(s)
- Endpoint:
- short-term toxicity to fish
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-02-27 to 2017-03-17, with definitive exposure phase from 2017-03-13 to 2017-03-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 203 (Fish, Acute Toxicity Test)
- Version / remarks:
- 1992
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.1 (Acute Toxicity for Fish)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Analytical monitoring:
- yes
- Details on sampling:
- Determination of the test item
Each test concentration and the control were analytically verified via LC-MS/MS from freshly prepared media after 0 and 72 hours and from corresponding aged test media after 24 and 96 hours. The analytical method validation was done according SANCO/3029/99 rev.4 (2000).
- Concentrations: 13.3 - 26.5 - 53 – 106 - 212 mg/L corresponding to a solid content of 6.28 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Sample storage conditions before analysis: The control and the samples were stabilized factor 2 with methanol after sampling. Further dilutions were made with methanol: fish dilution water (50:50) prior to analysis. All samples were stored at room temperature until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis. - Vehicle:
- no
- Details on test solutions:
- Test method
A semi-static test with daily renewal of the test media was performed.
Test concentrations
13.3 – 26.5 – 53 – 106 – 212 mg/L (factor 2) (nominal)
corresponding to a solid content of 6.28 – 12.5 – 25.0 – 50.0 – 100 mg/L. The concentrations were based on a preliminary range finding test and a first definitive study under threshold approach.
Preparation of the test medium
Appropriate amounts of the test item were weighed and transferred into the test vessels filled with appropriate volumes of dilution water. The test media were mixed with an ultraturrax (1 min, 17000 rpm). This procedure was repeated for every water renewal.
Control
7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates. - Test organisms (species):
- Danio rerio (previous name: Brachydanio rerio)
- Details on test organisms:
- TEST ORGANISM
- Common name: zebrafish
- Source: All fish used in the test was gained at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, 12307 Berlin, Germany)
- Length at study initiation: Average body length at the test start: 2.83 cm
- weight at study initiation: Average body weight at the test start: 0.23 g
ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions: 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. 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.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Acidity: 0.20 mmol/L (recent measurement 2017-01-10)
Alcalinity: 1.00 mmol/L (recent measurement 2017-01-10)
Conductivity: 181 µS/cm (recent measurement 2017-01-10)
- Type and amount of food during acclimation: The amount of food was 4 % of the fish body weight per feeding day. The test fish were not be fed 24 h before test start. Food: Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany
- Feeding frequency during acclimation: Food was provided 3 times per week.
- Health during acclimation (any mortality observed): No mortality was observed during this time. No disease treatments were administered throughout holding and testing.
FEEDING DURING TEST
Fish were not fed during the test. - Test type:
- semi-static
- Water media type:
- freshwater
- Remarks:
- tap water
- Limit test:
- no
- Total exposure duration:
- 96 h
- Post exposure observation period:
- not reported
- Hardness:
- 10 - 250 mg CaCO3/L. Total Hardness at day 0.59 mg CaCO3/L.
- Test temperature:
- 23 ± 2 °C, constant in a range of ± 1 °C during the test
- pH:
- 6.0 - 8.5
- Dissolved oxygen:
- ≥ 72 %, not less than 60 % of air saturation value.
- Salinity:
- not measured, freshwater
- Conductivity:
- 181 µS/cm
- Nominal and measured concentrations:
- Nominal: 13.3 - 26.5 - 53 – 106 - 212 mg/L corresponding to a solid content of 6.28 - 12.5 - 25.0 - 50.0 - 100 mg/L
For measured concentrations, please refer to section "any other details on results incl. tables". - Details on test conditions:
- TEST SYSTEM
- Test vessel: aquaria
- Type: loosely covered with a glass lid.
- Material, size, headspace, fill volume: 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)
- Aeration: No aeration was provided.
- Renewal rate of test solution: daily
- No. of organisms per vessel: Fish density in the test vessels was 0.16 g fish per litre test solution. 7 zebrafish were used for each test concentration and the control.
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): one
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Appropriate amounts of the test item were weighed and transferred into the test vessels filled with appropriate volumes of dilution water (same as for holding). The test media were mixed with an ultraturrax (1 min, 17000 rpm). This procedure was repeated for every water renewal.
- Chlorine: The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
- Alkalinity: 1.00 mmol/L
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all test vessels at 0, 24, 48, 72 and 96 hours
OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: natural photoperiod
- Light intensity: 7-750 Lux
EFFECT PARAMETERS MEASURED: Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 3, 24, 48, 72 and 96 h. ≥
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Control and 21.2 mg/L nominal test item concentration
- Test concentrations: 13.3 - 26.5 - 53 – 106 - 212 mg/L corresponding to a solid content of 6.28 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Results used to determine the conditions for the definitive study: The concentrations were based on a preliminary range finding test and a first definitive study under threshold approach. The threshold approach was carried out with the nominal concentration of 212 mg/L (100 mg/L solid content). As this test resulted in 86 % mortality of the fish within 24 h, first a preliminary test and thereafter a concentration-response test was carried out. - Reference substance (positive control):
- no
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC0
- Effect conc.:
- 53 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC100
- Effect conc.:
- > 212 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC50
- Effect conc.:
- > 212 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- not determinable
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC0
- Effect conc.:
- ca. 25 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: nominal concentration analytically verified
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC100
- Effect conc.:
- > 100 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: nominal concentration analytically verified
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC50
- Effect conc.:
- ca. 85 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: nominal concentration analytically verified
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC50
- Remarks:
- 24 to 96 hours
- Effect conc.:
- 180 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 95% CI: 106 - 212 mg/L
- Key result
- Duration:
- 96 h
- Dose descriptor:
- LC50
- Remarks:
- 24 to 96 hours
- Effect conc.:
- 85 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- mortality (fish)
- Remarks on result:
- other: 95% CI: 50.0 - 100
- Details on results:
- - Behavioural abnormalities: please see table 1 in section “any other details on results incl. tables”.
- Observations on body length and weight: not described
- Mortality of control: none
- Other adverse effects control: not reported
- Abnormal responses: please see table 1 in section “any other details on results incl. tables”.
- Effect concentrations exceeding solubility of substance in test medium: not applicable - Results with reference substance (positive control):
- not applicable (no reference item is recommended for this test according to the guidelines)
- Sublethal observations / clinical signs:
Table 1: Observations in the definitive test (n=7)
Nominal test item concentrations [mg/L]
Observation
Number of fish affected after different exposure periods [hours]
3
24
48
72
96
212
Exitus lethalis
0
4
0
0
0
hyperventilation
0
3
3
3
3
Missing escape reflex
0
0
3
0
0
Slow escape reflex
7
0
0
3
3
Side position
0
3
0
0
0
Normal behaviour
0
0
0
0
0
106
Exitus lethalis
0
2
0
0
0
Missing escape reflex
0
5
5
0
0
Slow escape reflex
7
0
0
5
5
53
Slow escape reflex
7
0
0
0
0
Normal behaviour
0
7
7
7
7
26.5
Normal behaviour
7
7
7
7
7
13.3
Normal behaviour
7
7
7
7
7
Control
Normal behaviour
7
7
7
7
7
Table 2: measured concentrations and percent of the nominal concentrations of the solid content of the test item
Fresh media, 0 hours
Old media, 24 hours
Fresh media, 72 hours
Old media, 96 hours
Test item [mg/L], nominal
Solid content, [mg/L], nominal
Measured conc. [mg/L]
%
Measured conc. [mg/L]
%
Measured conc. [mg/L]
%
Measured conc. [mg/L]
%
212
100
102
102
112
112
96.7
97
113
113
106
50.0
50.5
101
56.0
112
50.4
101
55.0
110
53.0
25.0
24.8
99
25.0
100
25.0
100
28.3
113
26.5
12.5
11.7
93
12.0
96
10.8
86
14.0
112
13.3
6.28
5.31
85
5.24
83
6.11
97
6.99
111
control
< LOQ
< LOQ
< LOQ
< LOQ
- Validity criteria fulfilled:
- yes
- Conclusions:
- In this study, the test item was found to cause lethal effects to zebrafish after 96 hours at a nominal test item concentration of 106 mg/L and higher. The LC50 after 96 hours was 180 (106 – 212) mg/test item/L, corresponding to 85.0 (50.0 – 100) mg solid content/L. The LC0 and LC100 after 96 hours was 53.0 and >212 mg test item/L, corresponding to 25.0 and >100.0 mg solid content/L (nominal concentrations).
- Executive summary:
The acute toxicity of the test item to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008) from 2017-02-27 to 2017-03-17, with a definitive exposure phase from 2017-03-13 to 2017-03-17 at the test facility.
An acute toxicity test under semi-static conditions with daily renewal of the test media was conducted with the nominal test item concentrations of 13.3 - 26.5 - 53.0 - 106 - 212 mg/L (factor 2), corresponding to a nominal solid content of 6.28 – 12.5 – 25.0 – 50.0 – 100 mg/L. Duration of the test was 96 hours. Seven test organisms were exposed to each test concentration and the control. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The determination of the test concentrations was carried out via LC‑MS/MS. Evaluation was done from freshly prepared media after 0 and 72 hours and from the corresponding 24 hours aged test media after 24 and 96 hours. The measured concentrations in freshly prepared media were in the range of 85 and 102 % of the nominal values. In corresponding 24 hours aged test media, the measured concentrations were between 83 and 113 % of the nominal values. Therefore, all effect values are based on nominal
concentrations of the test item. All validity criteria of the test guideline were met. The results of the study are summarized in the table below.
LC-Values (3 – 96 hours) of the Test Item
Based on nominal concentrations of the test item and the solid content [mg/L]
Test duration [h]
Test item [mg/L]
Solid content [mg/L]
LC50
Confidence interval
LC50
Confidence interval
3
> 212
n.a.
>100
n.a.
24
180
(106 – 212)
85.0
(50.0 – 100)
48
180
(106 – 212)
85.0
(50.0 – 100)
72
180
(106 – 212)
85.0
(50.0 – 100)
96
180
(106 – 212)
85.0
(50.0 – 100)
LC100=
Lowest test item concentration with 100 % mortality after 96 h
> 212
> 100
LC0=
Highest test item concentration with 0 % mortality after 96 h
53.0
25
Reference
Description of key information
The LC50 after 96 hours was 180 (106 – 212) mg/test item/L, corresponding to 85.0 (50.0 – 100) mg solid content/L.
The LC0 and LC100 after 96 hours was 53.0 and >212 mg test item/L, corresponding to 25.0 and >100.0 mg solid content/L (nominal concentrations).
Key value for chemical safety assessment
Fresh water fish
Fresh water fish
- Effect concentration:
- 85 mg/L
Additional information
The acute toxicity of the test item to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008) from 2017-02-27 to 2017-03-17, with a definitive exposure phase from 2017-03-13 to 2017-03-17 at the test facility.
An acute toxicity test under semi-static conditions with daily renewal of the test media was conducted with the nominal test item concentrations of 13.3 - 26.5 - 53.0 - 106 - 212 mg/L (factor 2), corresponding to a nominal solid content of 6.28 – 12.5 – 25.0 – 50.0 – 100 mg/L. Duration of the test was 96 hours. Seven test organisms were exposed to each test concentration and the control. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The determination of the test concentrations was carried out via LC‑MS/MS. Evaluation was done from freshly prepared media after 0 and 72 hours and from the corresponding 24 hours aged test media after 24 and 96 hours. The measured concentrations in freshly prepared media were in the range of 85 and 102 % of the nominal values. In corresponding 24 hours aged test media, the measured concentrations were between 83 and 113 % of the nominal values. Therefore, all effect values are based on nominal
concentrations of the test item. All validity criteria of the test guideline were met. The results of the study are summarized in the table below.
LC-Values (3 – 96 hours) of the Test Item
Based on nominal concentrations of the test item and the solid content [mg/L]
Test duration [h] |
Test item [mg/L] |
|
Solid content [mg/L] |
|
|
LC50 |
Confidence interval |
LC50 |
Confidence interval |
3 |
> 212 |
n.a. |
>100 |
n.a. |
24 |
180 |
(106 – 212) |
85.0 |
(50.0 – 100) |
48 |
180 |
(106 – 212) |
85.0 |
(50.0 – 100) |
72 |
180 |
(106 – 212) |
85.0 |
(50.0 – 100) |
96 |
180 |
(106 – 212) |
85.0 |
(50.0 – 100) |
LC100= Lowest test item concentration with 100 % mortality after 96 h |
> 212 |
|
> 100 |
|
LC0= Highest test item concentration with 0 % mortality after 96 h |
53.0 |
|
25 |
|
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
