Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to current test guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Reaction Product of Saccharose, Glycerine, biodiesel propoxylated
- Physical state: Red to brown, clear, liquid
- Analytical purity: 100%
- Lot/batch No.: T41/012/13
- Expiration date of the lot/batch: 2014-06-15
- Storage condition of test material: Ambient (room temperature)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sample storage conditions before analysis: All samples were stored at room temperature until the start of the
analysis, if necessary. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
An appropriate amount of the test item was weighed out and
transferred to an aquaria filled with an appropriate amount of
dilution water. The test item dispersions were treated with an
ultraturrax for 15 minutes (8500 rpm). After 24 ± 1.5 hours of
standing during strong aeration, the test dispersion were directly
used for exposure without filtration. Filtration of these solutions
would lead to organisational problems due to the high volumes
needed during exposure.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Forellenzucht Kasselmann, Forellental 12, 49170 Hagen, Germany. All fish used in the test were originated from the same delivery of the
supplier
- Length at study initiation (length definition, mean, range and SD): Average body length at the start of the exposure: 8. 7 cm.
- Weight at study initiation (mean and range, SD): Average body weight at the start of the exposure: 6.98 g.
- Feeding during test: Fish were not fed during the period of the test.


ACCLIMATION
- Acclimation period: Only rainbow trout with at least 12 days of acclimatization in the
laboratory and mortality< 5 % within the last 7 days before the study
start were used in the test. At least 7 days before testing, the fish
were held in water of the quality to be used in the test.
- Type and amount of food: Forellenfutter Zuchtfutter 0.8 - 1.2 mm (supplier: TEICHCENTERRIEGER,
Nudlingstr. 13, 63607 Wachtersbach-Aufenau, Germany;
producer: COPPENS INTERNATIONAL Bv, P.O. Box 534, 5700 AM
Helmond, the Netherlands). The amount of food was 1.5 % of the fish body weight per feeding day. The test fish were not fed 24 h before
the start of the exposure.
- Feeding frequency: Food was provided daily.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h

Test conditions

Hardness:
10 - 250 mg CaCQ3/L
Test temperature:
13 - 17 °C, controlled at± 1 °C.
pH:
6.0 - 8.5
Dissolved oxygen:
Not less than 60 % of air saturation value.
Nominal and measured concentrations:
nominal: 4.27, 9.39, 20.7, 45.5, 100 mg/L
measured: The measured test item concentration in the
fresh media was in the range of 97 to 119 % (0 h) and 96 to 118 % (72 h). In the 24 h old media,
the measured test item concentration was in the range of 103 to 118 % (24 h) and 84 to 115 %
(96 h).
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass aquaria of 64 L were used, filled with 55 L of dilution water
(dimensions: 40/40/40 cm, depth of water: 35 cm), and covered
with glass plates
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: About 55 L per vessel.
- Aeration: Gentle 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 loading level and control.
- No. of vessels per concentration (replicates): One replicate per loading level and control.
- Biomass loading rate: fish density in the tanks was 0.888 g fish per liter test solution

TEST MEDIUM / WATER PARAMETERS
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.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCQ3/L
pH-value: 6.0 - 8.5

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16 hours photoperiod occurred during the exposure


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : pH-value, temperature and oxygen saturation were measured
in all test vessels at the beginning and at the end of the
exposure and every 24 hours in the new and old media. In the
control, total hardness of the water was determined at the beginning
of the exposure. During exposure, the water temperature was
recorded continuously by a data logger. The light intensity on the
surface of the test aquaria was measured at the start of the
exposure. Observations were made after 4, 24, 49, 75 and 96 hours of
exposure

TEST CONCENTRATIONS

- Range finding study: The loadings were based on the results of non GLP preliminary
range finding tests
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
48.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LL0
Effect conc.:
20.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LL100
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Recommended average total length at the start of the exposure
should be 6.0 ± 2.0 cm (acc. to EU guideline). Fish with the
recommended length were not available during this season. For
this reason, fish with a higher length were used.
This deviation is considered to have no impact on quality and
integrity of the study.

Applicant's summary and conclusion