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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5.10.84 - 15-10-84
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. The study was performed with test material being used to support the substance on the basis of read-across.
Qualifier:
according to
Guideline:
other: German Federal Environment Agency [UBA] - Proposed procedure 'Lethal effect on zebra danio (Brachydanio rerio)
Deviations:
yes
Remarks:
(excessive loss of test material in the basic suspension even though this was replaced with fresh solution twice daily)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: approximately (1-2) x 10⁻⁴ mbar at 20 °C
- Water solubility: 4-6 mg/L
- Stability in water: Hydrolytically stable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Analytical determinations were presented for 0, 4, 6.1 and 15.1 mg/L and the basic suspension (200 mg/L) was also analysed.
- Sampling method: The concentration of the test material was analysed at 0 hours (test solutions replaced with fresh solution), 48 hours (test solution was 15 hours old at sampling) and 96 hours (test solution was 15 hours old at sampling).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An emulsion of the test material was prepared in the test medium. The test material was ultrasonicated for 30 minutes to create a test concentration of 2000 mg/L. This suspension was diluted the test medium to 200 mg/L and stored. Before use, the contents of the storage containers were thoroughly mixed with an Ultra Turrax disperser for 90 seconds and simultaneously circulated by the test system pump.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Source: Westaquarium

ACCLIMATION
- Health during acclimation (any mortality observed): 0 % mortality
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
2870 m Mol/m³ (16.0 °d)
Test temperature:
22-23.5 °C
pH:
7.6-7.8
Dissolved oxygen:
6.4-7.7 mg/L (≥76.3-91.9 % saturation based on 23 °C)
Nominal and measured concentrations:
Nominal concentrations: 0, 2.1, 3.0, 4.0, 6.1, 9.0, 12.0 and 15.1 mg/L
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 2 L fish tanks
- Type: open
- Renewal rate of test solution: The basic solution was replaced with fresh solution twice a day. The tanks undergo four replacements over 24 hours.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2 tanks (A and B)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dusseldorf portable water

EFFECT PARAMETERS MEASURED: Fish were observed for mortality and sublethal effects at 0, 6, 24, 48 and 96 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
not corrected for test material purity
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
not corrected for test material purity
Basis for effect:
mortality
Remarks on result:
other: ± 22.5 %
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
6.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
not corrected for test material purity
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
0.63 - 2.1 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Corrected nominal values to account for loss of test material
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.13 - 3.78 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Remarks:
not corrected for test material purity
Basis for effect:
mortality
Remarks on result:
other: Corrected nominal values to account for loss of test material
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
1.83 - 6.1 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Remarks:
not corrected for test material purity
Basis for effect:
mortality
Remarks on result:
other: Corrected nominal values to account for loss of test material
Details on results:
- Behavioural abnormalities: Sublethal effects could only be determined during the first hours of testing and only with concentrations >LC0 (96 hours). No mortality was seen in the controls.
- Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Excessive loss of the test material via evaporation was unavoidable as it occurred largely in the basic suspension.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- LC50: 150 mg/L
- Other: LC0 90 mg/L; LC100 250 mg/L
Reported statistics and error estimates:
The LC50 of the test material was calculated by probit analysis.
During the test, the basic solution was renewed every 24 hours. Nevertheless, there was still a considerable loss of the test substance. The analysis values reveal that the equipment dosed properly and evenly but that the content in the basic suspension dropped significantly between two new preparations.

In the basic suspension to approximately 60 % of the nominal value and in the test tank in the range of LC0 to LC100 to approximately 30 % of the nominal value. This high loss of test material is caused by high volatility. The test results after 0 hours revealed that the method of analysis has a recovery rate of 100 % and that the test system dosed properly into the test tank because the nominal values reached >92 % of the target.

Loss via evaporation was unavoidable, as it largely occurred in the basic suspension and fresh preparations could not be achieved more frequently than twice a day. This could only be remedied if the entire system was hermetically sealed, which was not possible. In order to maintain the effective concentration, the LC50 values were multiplied by a factor of 0.3 (the results were therfore presented as a range).

Table 1. Results

   % mortality for each 10 fish            
 Conc. in mg/L  0 hours  6 hours  24 hours  48 hours  96 hours
 2.1  0  0  0  0  0
 3.0  0  0  30  30  30
 4.0  0  0*  40  50  50
 6.1  0  10*  90  100  100
 9.0  0  40*  100  100  100
 12.0  0  100*  100  100  100
 15.1  0  80*  100  100  100
 LC0  -  4.0  2.1  2.1  2.1
 LC50  -  -  -  3.78 ± 22 %  3.78 ± 22.5 %
 LC100  -  12.0  9.0  6.1  6.1

*sub-lethal symptoms: lethargic, timid (pressed to the ground), difficulties with equilibrium

Table 2: Analytical results

 Nominal conc (mg/L)  Acutal value / % of nominal value      
   0 hours¹  48 hours²  96 hours²
 Fish tank         
 0  unknown / -  unknwon / -  unknown / -
 4  4.1 / 102  1.34 / 33.5  1.25 / 31.3
 6.1  5.9 / 96.7  2.36 / 38.7  2.20 / 36.1
 15.1  13.84 / 91.7  7.9 / 52.3  5.75 / 38.1
 Basic suspension         
 200 mg/L²  202 / 101  235 / 117.5  206 / 103
 200 mg/L²  - / -  126.1 / 63.1³  119.0 / 59.5³

¹ Replaced with fresh solution

² 15 hours old (8 am after 15 hours operation with the same basic suspension)

³ Without the values 1117.5 and 103, loss increased with descending test concentrations.

Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the test, the 96 h LC50 of the test material to zebra fish has been determined to be between 1.13 and 3.78 mg/L (nominal concentration 3.8 mg/L).
Executive summary:

A short term toxicity test performed in compliance with the guideline German Federal Environment Agency [UBA] - Proposed procedure 'Lethal effect on zebra danio (Brachydanio rerio) which was in basic compliance with the OECD Guideline 203 and performed under GLP conditions investigated the short term toxicity of the test material cyclohexyl salicylate in fish. Zebra fish (Danio rerio) were exposed to the test material at nominal concentrations of 0, 2.1, 3.0, 4.0, 6.1, 9.0, 12.0 and 15.1 mg/L under flow through conditions. Sublethal effects were noted in the fish at 6 hours at and above 4.0 mg/L. Sublethal symptoms included lethargy, timidity and difficulties with equilibrium. Due to the high volatility of the test material, the relevant lethal concentrations (LC0, LC50 and LC100) were adjusted using a factor of 0.3 to account for the loss via evaporation. The results were therefore presented as a range. Under the conditions of the test, the 96 h LC50 of the test material to zebra fish was determined to be between 1.13 and 3.78 mg/L (nominal concentration 3.8 mg/L).

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please see 'Justification for read-across to support the REACH registration of (z)-3-hexenyl salicylate' document attached (section 13) for full details.

It is proposed to read-across to another salicylate substance in order to fulfil data requirements.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please see ''Justification for read-across to support the REACH registration of (z)-3-hexenyl salicylate' document attached for full details.

Source substance: cyclohexyl salicylate (EC 400-410-3, CAS 25485 88-5).
Target substance: (z)-3-hexenyl salicylate (EC 265-745-8, CAS 65405-77-8)

3. ANALOGUE APPROACH JUSTIFICATION
Please see 'Justification for read-across to support the REACH registration of (z)-3-hexenyl salicylate' document attached for full details.

(z)-3-hexenyl salicylate (the target substance) and the read-across substance cyclohexyl salicylate (source substance) have been characterised using the categories and databases present in the OECD QSAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. The main difference observed is structural. The target substance ((z)-3-hexenyl salicylate) contains an acyclic hexenyl chain and the source substance (cyclohexyl salicylate) contains a cyclic hexyl group.
The output from the OECD QSAR Toolbox shows that the profiles of (z)-3-hexenyl salicylate and cyclohexyl salicylate are sufficiently similar such that any available data from the source substance can be used to address the following endpoints in the REACH registration dossier for (z)-3-hexenyl salicylate:

4. DATA MATRIX
Please see 'Justification for read-across to support the REACH registration of (z)-3-hexenyl salicylate' document attached for full details.
Reason / purpose:
read-across source
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
6.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the test, the 96 h LC50 of the test material to zebra fish has been determined to be between 1.13 and 3.78 mg/L (nominal concentration 3.8 mg/L).

Description of key information

The acute toxicity 96 hour LC50 in fish was determined to be between 1.13 and 3.78 mg/L; study conducted in accordance with sound scientific principles; Gode and Gerike, 1984

Key value for chemical safety assessment

LC50 for freshwater fish:
3.78 mg/L

Additional information

The acute toxicity of cyclohexyl salicylate (a structural analogue of (Z)-3-hexenyl salicylate ) was assessed in zebra fish in the key study. The key study was performed in compliance with the guideline German Federal Environment Agency [UBA] - Proposed procedure 'Lethal effect on zebra danio (Brachydanio rerio) which was in basic compliance with the OECD Guideline 203 and performed under GLP conditions investigated the short term toxicity of the test material cyclohexyl salicylate in fish. Zebra fish (Danio rerio) were exposed to the test material at nominal concentrations of 0, 2.1, 3.0, 4.0, 6.1, 9.0, 12.0 and 15.1 mg/L under flow through conditions. Sublethal effects were noted in the fish at 6 hours at and above 4.0 mg/L. Sublethal symptoms included lethargy, timidity and difficulties with equilibrium. Due to the high volatility of the test material, the relevant lethal concentrations (LC0, LC50 and LC100) were adjusted using a factor of 0.3 to account for the loss via evaporation. The results were therefore presented as a range. Under the conditions of the test, the 96 h LC50 of the test material to zebra fish was determined to be between 1.13 and 3.78 mg/L (nominal concentration 3.8 mg/L). The study was performed in line with good scientific principles and reported in sufficient detail to assess the results and conclusions of the study. The study was performed on a structural analogue, which is deemed sufficiently structurally and mechanistically to use the results of the present study to read-across to (Z)-3 -hexenyl sailcylate to fulfil the data requirement.

The acute toxicity of (Z)-3 -hexenyl salicylate was assessed in orange killifish in the supporting study. The study was non-GLP and performed to sound scientific principles but the study duration was 48 hours and therefore less suitable to assess the acute toxicity of the substance for classification and labelling and risk assessment purposes. The substance is readily biodegradable and so a semi-static or flow-through test system should have been used and no analytical measurements of the test material concentration were made. The results, however, concur with the results and conclusions of the key study.