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Ecotoxicological information

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:
2008-11-10 to 2008-11-14
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
GLP compliance:
yes (incl. QA statement)
Remarks:
, signed 2006-06-01
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
5 dilution levels 6.25 – 12.5 – 25 – 50 – 100 % (factor 2) saturated solution and the control were analytically verified from freshly prepared media after 0, 24, 48 and 72 h and from 24 h old media after 24, 48, 72 and 96 h. The analytical method was validated.

- Sample storage conditions before analysis:
All samples were stored at room temperature until start of analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A saturated solution (Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6) of 10 mg test item/L was daily prepared with solution a slow-stirring procedure in a closed bottle. Twice 40 µL test item/3L test medium were stirred for 24 h at 20 °C ± 2 °C in a brown glass bottle. After phase separation (drop formation on the water surface) appropriate amounts of the saturated solution (= bottom phase) and dilution water were filled into the test vessels.

The concentrations were based on the results of a preliminary static range finding test.
Test Concentrations:
6.25 – 12.5 – 25 – 50 – 100 % (factor 2) saturated solution

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: Aquarium am Aegi, Volgersweg 6, 30175 Hannover, Germany
- Length at study initiation: average body length at the test start: 1.84 cm
- Weight at study initiation: average body weight at the test start: 0.043 g
- Feeding during test: the test fish were not fed 24 hours before the test was started and during the test.

ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions: same as test (23 +- 2 °C, diffuse light and dissolved oxygen concentration > 80 % )
- Type of food: Stör perlets; SERA GmbH, 52518 Heinsberg, Germany
- Amount of food: The amount of food was 4 % of the fish body weight per feeding day.
- Feeding frequency: Food was provided 3 times per week. The test fish were not fed 24 h before the test started.
- Health during acclimation (any mortality observed): fish with a mortality < 5 % within these days before start of the study were used in the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
not applicable
Hardness:
63 to 77 mg CaCO3/L
- measurements were taken at 0, 24, 48, 72 hours
- value within acceptable limits
Test temperature:
21.3 to 22.5 °C
- measurements were taken at 0, 24, 48, 72 and on termination of the test after 96 hours
-all values within acceptable limits
pH:
7.10 to 7.36
- measurements were taken at 0, 24, 48, 72 and on termination of the test after 96 hours
- all values within acceptable limits
Dissolved oxygen:
84 to 100 %
- measurements were taken at 0, 24, 48, 72 and on termination of the test after 96 hours
- all values within acceptable limits
Salinity:
not applicable
Nominal and measured concentrations:
The concentrations were based on the results of a preliminary static range finding test.
5 test concentrations in a geometrical series with a dilution factor of 2:
6.25 – 12.5 – 25 – 50 – 100 % saturated solution, corresponding to
0.326 – 0.374 – 1.63 – 4.27 – 7.10 mg/L geometric mean measured test item concentration.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass-aquaria loosely covered by glass tops (with reduced headspace) were used.
- Material, size, headspace, fill volume: volume of about 2 L per vessel
- Aeration: No aeration was provided.
- Renewal rate of test solution (frequency/flow rate): daily renewal of the test media
- No. of organisms per vessel: 7 zebrafish were used per test concentration and control
- No. of vessels per concentration (replicates): One replicate per test concentration
- No. of vessels per control (replicates): One replicate per control.
- Biomass loading rate: fish density in the tanks was less than 1 g fish per litre test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all vessels after beginning the test and every 24 h (from old and new media). Total hardness of the water was determined at the beginning out of the test control and after any renewal. During the test the room temperature was recorded continuously with a thermohygrograph.

OTHER TEST CONDITIONS
- Photoperiod: natural photoperiod
- Light intensity: 0.1-10 µmol photons • m-2 • s-1,

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations were made after 24, 48, 72 and 96 h. 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.).

PREMILINARY TEST
- Range finding study: yes, non-GLP, 5 fish were used per concentration.
- Test concentrations: saturated solution and 10 % saturated solution
- Spacing factor for test concentrations: 2
- Results used to determine the conditions for the definitive study: The results showed no mortalities at the 10 % saturated solution and 100 % mortality at the saturated solution. Based on this information, the test concentrations, in duplicate, were selected for the definitive test.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
2.64 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
mortality (fish)
Remarks on result:
other: 95 % CL: 1.63 - 4.27 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.326 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
other: mortality and behaviour
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
1.63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
4.27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
mortality (fish)
Details on results:
100 % cumulative mortality occurred at the 7.10 mg/L solution and at the 4.27 mg/L test item concentration. 0 % cumulative mortality occurred at 1.63, 0.374 and 0.326 mg/L. Behavioral abnormalities occurred at 1.63 and 0.374 mg/L. For more information of symptoms of toxicity during the test at each time of observation phase, please refer table 3 of "Any other information on results incl. tables".

- Mortality of control: none of the fishes in the control group died.

The test item concentration decreased within 24 hours to 12 – 90 % of the measured initial values. All effect levels were based on geometric mean measured values.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
After 24 - 96 h only 0 and 100 % mortality was found. No evaluation regression analysis was carried out. Hence the LC50 is the geometric mean value of the concentrations with 0 and 100 % mortality. The concentration causing 0 and 100 % mortality were chosen as lower and upper confidence limit. The concentrations causing to 0 and 100 % mortality (LC0 and LC100) as well as the no effect level (NOEC) after 96 h were determined directly from the test results. All effect levels were based on geometric mean measured values.
Sublethal observations / clinical signs:

Range Finding Test (non GLP)


Biological Data


Table 1:           Cumulative Mortality in thePreliminary Test


              (n = 5)

































Nominal test item concentration [mg/L]



Test Duration [hours]



24



48



72



Saturated solution



100



100



100



10 % saturated solution



0



0



0



Control



0



0



0



 


Measured Concentrations


Table 2:           Measured concentrationsin the Preliminary Test


              (measured after storage of 4 weeks at 6 ± 2 °C)





























Nominal test item concentration [mg/L]



test item



0 h


Meas. Conc.


[mg/L]



24 h


Meas. Conc.


[mg/L]



Saturated solution



1.32



0.088



10 % saturated solution



< LOQ



< LOQ



Control



< LOQ



< LOQ



Meas. Conc.    = Measured concentration, enrichment and dilution factor taken into account


 


Definitive Test


Biological Data


Table 3:           Observations in the Test Vessels


                       (n=7)





























































































Geometric mean measured test item concentration [mg/L]



Effect*



                        Test Duration [h]                                



24



48



72



96



7.10



(E)



7/7



---



---



---



4.27



(E)



7/7



---



---



---



1.63



(2.1)



3/7



-



-



-



(2.4)



-



-



3/7



7/7



(2.5)



7/7



7/7



4/7



-



(2.6)



3/7



5/7



-



-



  0.374



(1)



-



-



-



7/7



(2.4)



7/7



7/7



7/7



-



  0.326



(1)



7/7



7/7



7/7



7/7



Control



(1)



7/7



7/7



7/7



7/7



*) The number in brackets correspond to the following observation:


(1)      = Normal behaviour    (2.5)    = No escape reflex
(2.1)    = Lethargy                    (2.6)    = No swim-up behaviour
(2.4)    = Slow escape reflex  (E)       = Exitus lethalis
-          = No Observations      ---       = No Observations due to 100 % mortality


 


  Table 4: Overview of Measured Concentrations and Mean Measured Concentrations of the test substance


 



































































































Dilution level of the saturated solution



1st Sampling
(day 0 / 1)
Meas. conc.

[mg/L]



2nd Sampling
(day 1 / 2)
Meas. conc.

[mg/L]



3rd Sampling
(day 2 / 3)
Meas. conc.

[mg/L]



4th Sampling
(day 3 / 4)
Meas. conc.

[mg/L]



Geometric
Mean
Meas.
Conc.



 



0 h



24 h



0 h



24 h



0 h



24 h



0 h



24 h



[mg/L]



 1:1



8.62



 5.85



   −













7.10



 1:2



4.65



 3.92



   −













 4.27



 1:4



2.96



 1.24



2.75



1.10



1.50    



1.35



2.44   



0.888



 1.63



 1:8



0.799



 0.151



0.310



0.198



0.991



 0.495



0.925



 0.114



 0.374



  1:16



0.520



 0.134



0.387



0.217



0.602



 0.272



0.673



 0.200



 0.326



Control



< LOQM



< LOQM



< LOQM



< LOQM



< LOQM



< LOQM



< LOQM



< LOQM



< LOQM




 


Conc.                                   = Concentration


Meas. conc.                        = Measured concentration


LOQM                                  = Limit of quantification of the analytical method (0.06 mg/L)


−                                           = Not analysed due to 100 % mortality

Validity criteria fulfilled:
yes
Remarks:
The study was performed and met the validity criteria: O2 - saturation was ≥ 60 % , Mortality in the control was 0 %, results based on geometric mean measured concentrations of the test item, for constant conditions semi-static procedure
Conclusions:
The 96-hours LC50 (geometric mean measured test item concentration, 0 - 96 h) was 2.64 mg/L (1.63 - 4.27 mg/L), the 96-hours NOEC was 0.33 mg/L (geometric mean measured test item concentration).
Executive summary:

The toxic effect of the test item to Danio rerio according to OECD 203 was assessed in a semi-static dose-response test in a loosely covered glass-aquaria. For the determination of the test concentrations one range-finding test was performed. The test concentrations were verified by GC-FID analysis.At the end of the test the toxicity of the test item to Danio rerio had increased. After 96 hours of exposure no mortality and no effects on behaviour of the test animals was observed in the controls and in the measured (geometric mean) test item concentration of 0.33 mg test item/L.


 


At the measured (geometric mean) concentration of 4.27 mg test item/L, 7 out of 7 died (LC100).


 


The 96-hour LC50was determined to be 2.64 mg test item/L based on geometric mean measured concentration.

Description of key information

LC50 (96 h) 2.64 mg/L (measured, geom. mean value)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
2.64 mg/L

Additional information

The toxic effect of the test item to Danio rerio according to OECD 203 was assessed in a semi-static dose-response test in a loosely covered glass-aquaria. For the determination of the test concentrations one range-finding test was performed. The test concentrations were verified by GC-FID analysis. At the end of the test the toxicity of the test item to Danio rerio had increased. After 96 hours of exposure no mortality and no effects on behaviour of the test animals was observed in the controls and in the measured (geometric mean) test item concentration of 0.33 mg test item/L.

At the measured (geometric mean) concentration of 4.27 mg test item/L, 7 out of 7 died (LC100).

 

The 96-hour LC50 was determined to be 2.64 mg test item/L based on geometric mean measured concentration.