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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:
1989-12-04 to 1989-12-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP. However, there was no analysis of exposure concentrations and the solvent concentration was higher than recommended by the OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
The concentration of tertiary butyl alcohol (TBA) used in the preparation of the test media (320 μL/L) exceeded the maximum (100 mg/L) recommended in the OECD guideline.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Stock solutions of the test substance were prepared in tertiary butyl alcohol at concentrations of 100, 180 and 320 g/l. The stock solutions were added to 1.5 litres of dilution water in amounts required to produce the desired test concentrations. Fresh stock solutions and test media were prepared daily. The final concentration of TBA in the media (320 μl/l) exceeded the maximum concentration recommended in the OECD test guideline (100 mg/L).

- Controls: Dilution water and Dilution water+ Tertiary butyl alcohol at 320 μl/l.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): tertiary butyl alcohol (TBA).

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 320 μl/l.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Oily droplets were observed on the surface of the 100 mg/l test medium.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM

- Common name: Zebrafish

- Source: M.B. Ruysbroek B.V., Maassluis, The Netherlands.

- Length at study initiation (length definition, mean, range and SD): 2.3 +/-0.1 cm

- Weight at study initiation (mean and range, SD): 0.1 +/-0.01 g

- Feeding during test: none
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
215 mg/l as CaCO3
Test temperature:
25 +/-1ºC
pH:
7.6-8.1
Dissolved oxygen:
Generally ≥7.1 mg/l. A low concentration 3.9 mg/l was measured in the 58 mg/l test concentration but was it was not thought to be the cause of the death of the fish in this treatment.
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0+320 μl/l TBA (Solvent control), 10, 18, 32, 58 and 100 mg/l.

Test concentrations all contained 320 μl/l TBA
Details on test conditions:
TEST SYSTEM

- Test vessel: Beakers

- Type: open

- Material, size, fill volume: glass, 2 litre containing 1.5 litres of test medium

- Aeration: yes

- Renewal rate of test solution (frequency/flow rate): Daily

- No. of organisms per vessel: 10

- No. of vessels per concentration (replicates): 2

- No. of vessels per control (replicates): 2

- No. of vessels per vehicle control (replicates): 2

- Biomass loading rate: 0.67 g/l

TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: DSWL standard water prepared by adding salts to groundwater from Linschoten, the Netherlands

- Ca/mg ratio: 1.36 mmol/l Ca2+ : 0.73 mmol/l Mg2+

- Culture medium different from test medium: no

- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS

- Adjustment of pH: no

- Photoperiod: 16 h light, 8 h dark

EFFECT PARAMETERS MEASURED: mortality after 24, 48, 72 and 96 hours

TEST CONCENTRATIONS

- Spacing factor for test concentrations: 1.8

- Range finding study; yes but results not reported
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 10 - < 32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: calculated as the geometric mean of the concentration range bounding the LC50
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality of control: 0 in both dilution water control and in dilution water + TBA control
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: at 100 mg/L small oily droplets were observed after dosing.
Reported statistics and error estimates:
No statistical analysis of the test results was carried out

Table 1. Test results

 Nominal test substance concentration (mg/l)  Mean percentage mortality after 24 hours  Mean percentage mortality after 48 hours Mean percentage mortality after 72 hours  Mean percentage mortality after 96 hours 
 0 (Control)  0 0  0  0
 0 (TBA control)  0  0  0  0
 10  0  0  0  0
 18  0  5  85  85
 32  100  100  100  100
 58  100  100  100  100
 100  100  100  100  100

The authors of the study report determined the 96-h LC50 between 10-32 mg/l to be equivalent to 18 mg/l as the geometric mean of the concentration range.

However, the current reviewers are of the opinion that 18 mg/l is not correct because 85% mortality was determined at 18 mg/l. There are insufficient data to calculate the LC50 for this study and the test material contains a thiocyanate, considered to have a specific mode of action. Substances with a specific mode of action often have sharp dose-response curves, further increasing the uncertainty associated with the derivation of an LC50 from the data.

Validity criteria fulfilled:
yes
Conclusions:
A 96-hour LC50 value of between 10 and 18 mg/l, and a NOEC of 10 mg/l have been determined for the effects of the test substance on mortality of Danio rerio (tested as Brachydanio rerio). The results are interpreted with reference to nominal concentrations of the test substance. However the test substance is susceptible to hydrolysis and it is likely that the test organisms were primarily exposed to a mixture of the parent compound and the hydrolysis products of the substance.

Description of key information

96hr LC50: 10-18 mg/l (nominal)

Key value for chemical safety assessment

LC50 for freshwater fish:
10 mg/L

Additional information

A 96 hour LC50 value of between 10 and 18 mg/l and a NOEC of 10 mg/l have been determined for the effects of the test substance on mortality of Danio rerio (tested as Brachydanio rerio). The results are interpreted with reference to nominal concentrations of the test substance.

The test solution was prepared using tert-butyl alcohol (TBA) as a solvent. The recommended solvent concentration (100 mg/l) was exceeded during the test, with TBA at a concentration of approximately 250 mg/l (based on a density of 0.781 g/ml). However, according to the REACH registration dossier for TBA (2-methylpropan-2-ol), the lowest E(L)C50 value is 933 mg/l for invertebrates. It is therefore unlikely that TBA significantly affected the results of the test.

The authors of the study report determined the 96 hour LC50 to be between 10-32 mg/l and equivalent to 18 mg/l as the geometric mean of the concentration ranges.

However, the current reviewers are of the opinion that 18 mg/l does not represent the correct LC50 value because 85% mortality was determined at 18 mg/l, while 0% mortality was reported at the 10 mg/l concentration. There are insufficient data to calculate the LC50 for this study and the test material contains a thiocyanate functional group, considered to have a specific mode of action. Substances with a specific mode of action often have sharp dose-response curves, further increasing the uncertainty associated with the derivation of an LC50 from the data. For these reasons, the current reviewers are choosing to set a conservative LC50 at 10 mg/l for the purpose of deriving a PNEC.

In addition, the test substance is susceptible to hydrolysis and it is likely that the test organisms were exposed to a mixture of the parent compound and the hydrolysis products of the substance.

A functional group associated with ecotoxicity by a specific mode of action (thiocyanate) is present in both the registration substance and the silanol hydrolysis product, and the effects seen are attributed to this. It is therefore reasonable to conclude that both the parent and hydrolysis product could contribute to any effects observed. Daily renewal of test media was used and no undissolved material was observed at the test concentrations from which the LC50 is calculated (some oily droplets were observed after dosing at 100 mg/l). In the absence of analytical monitoring, it is not possible to estimate with confidence the concentrations of parent form or hydrolysis product in the test system.

It is therefore considered appropriate and conservative to use this result without adjustment as the starting point for deriving

·        PNECaqua and PNECsediment (for registration substance)

·        PNECsoil and PNECsediment (for silanol hydrolysis product, by Equilibrium Partitioning method)