Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
28 Oct. 2008 - 27 Jan. 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): wash oil
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Substance type: organic
- Physical state: liquid
- Stability under test conditions: WAF (water accommodated fraction) limited
- Storage condition of test material: room temperature, exclusion of light

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fractions (WAFs) prepared by stirring various amounts of the test item in dechlorinated tap water
for 24 h with magnetic stirrers. The WAFs were prepared in 20-L glass fish tanks (range-finding: 2-L glass beaker).
These were equipped with a glass tube that allowed the separation of the water phase by siphoning.
The test loadings of wash oil were weighed on weighing scoops, that afterwards were placed into the glass vessels.
The glass tanks or beakers were then filled with the tap water.
Mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction
of the test item. To ensure this, the vortex developed at the surface by stirring was set at ~ 10 % of the water depth.
After stirring for 24 h the WAFs were allowed to stand for 1 h before use to facilitate phase separation.
The extracts gained with this method were clear.

- Differential loading: control 10, 25, 50, 100, and 250 mg/L (main test)
- Controls: without test material (blank; tap water)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Strain:
- Source: Max-Planck-Institute für Entwicklungsbiologie, Tübingen
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): 3.0 - 3.5 cm
- Weight at study initiation (mean and range, SD): no data
- Feeding during test: none


ACCLIMATION
- Acclimation period: >12 days
- Acclimation conditions: same as test
- Type and amount of food: no data
- Feeding frequency: no data
- Health during acclimation (any mortality observed): mortality <1%

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
water/WAF renewal: every 24 h
Post exposure observation period:
none

Test conditions

Hardness:
Total hardness 8.97 °dH
Carbonate hardness 6.98 °dH
Test temperature:
21.1 - 22.0 °C (23 +-2 °C)
pH:
8.2 - 8.6
Dissolved oxygen:
81 - 101 % of saturation
Salinity:
no data
Nominal and measured concentrations:
nominal: 10, 25, 50, 100, and 250 mg/L
measured (as TOC):

Period Control 10 mg/L 50 mg/L 250 mg/L (loading)
====================================================
0-24 h start 3.0 3.9 6.2 23.7 mg/L (TOC of WAF)
0-24 h end 2.8 3.5 5.8 21.3 mg/L (TOC of WAF)
72-96 h start 2.7 4.3 5.7 - mg/L (TOC of WAF)
72-96 h end 2.7 4.3 5.3 - mg/L (TOC of WAF)
======================================================
The oil-related TOC values were approx. 0.7 - 1.5 mg/L (at 10 mg oil/L), 2.5 - 3.5 mg/L (at 50 mg oil/L),
and 20 mg/L (at 250 mg oil/L).
Details on test conditions:
TEST SYSTEM (main test)
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 20 L, test volume 7 L
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): every 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: 1 fish/L


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: light / dark: 14 h/ 10 h
- Light intensity: fluorescent tubes, no data


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Swarming-behaviour, activity, balance, surfacing, mortality, breathing frequency, apathy


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 - 2.5
- Range finding study
- Test concentrations: 10, 100, and 1000 mg/L
- Results used to determine the conditions for the definitive study: no mortality at 10 and 100 mg/L, 100 % at 1000 mg/L
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
79 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: ; (LL = lethal loading)
Duration:
96 h
Dose descriptor:
LL10
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
behaviour
Duration:
96 h
Dose descriptor:
LOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
behaviour
Remarks:
: slightly reduced activity from 48 h onwards
Details on results:

- Control: Swarming and swimming behaviour and activity were normal.

- 10 mg/L: Comparable to control during the observation periods of 0 – 24 h and 24 – 48 h.
Activity was slightly reduced during the observation periods 48 – 72 h and 72 – 96 h.
The animals partially used the lower part of the vessel for swimming, and surfacing could be observed.

- 25 mg/L: Activity was slightly reduced during the observation periods 0 – 24 h and 24 – 48 h.
Activity was reduced during the observation periods 48 – 72 h and 72 – 96 h. Surfacing could be observed
during all observation periods. One fish died during the observation period of 72 – 96 h.

- 50 mg/L: Activity was reduced from the observation periods of 0 – 24 h to 72 – 96 h. Surfacing could be observed
during all observation periods. One fish died during the observation period 24 – 48 h.

- 100 mg/L: Activity was reduced during the observation periods of 0 – 24 h and 24 – 48 h. One fish died during the
observation period of 24 – 48 h. Activity was strongly reduced during observation periods of 48 – 72 h and 72 – 96 h.
Surfacing could be observed during all observation periods. The breathing frequency of the fish was elevated,
and signs of imbalance could be observed. During the observation periods of 48 – 72 h and 72 – 96 h,
all fish were strongly affected, the animals lay mostly at the bottom of the test vessel and were not able to swim coordinately.
These sublethal effects could be observed on the three surviving fish until the end of the test.
Three other fish died during the observation periods 48 – 72 h and 72 – 96 h.

- 250 mg/L: The fish showed strong toxic effects shortly after the start of the observation period 0 – 24 h.
The breathing frequency of all fish was elevated, surfacing and imbalance could be observed.
All seven fish died during the observation period of 0 – 24 h.


Observations at the end of the test after feeding of the surviving fish:

- Control: All fish fed immediately.
- 10 mg/L: All fish fed with slight retardation compared to the control.
- 25 mg/L: The 6 surviving fish fed with retardation compared to the control.
- 50 mg/L: The 6 surviving fish did not feed.
- 100 mg/L: The 3 surviving fish did not feed.

After the end of the test, the surviving fish were kept in de-chlorinated tap water from 20.12.08 to 23.12.08.
During this period, fish derived from the control and the test loading rates of 10, 25 and 50 mg/L recovered and
showed normal behaviour. The three surviving fish derived from test concentration of 100 mg/L recovered somewhat
and were able to swim again, however these fish remained affected.

Any other information on results incl. tables

Number of dead fish and cumulative lethality [%] at various observation times (Report, Tab. 3)

Time [h]

Control

10 mg/L

25 mg/L

50 mg/L

100 mg/L

250 mg/L

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Number

[%]

24

0/7

0

0/7

0

0/7

0

0/7

0

0/7

0

7/7

100

48

0/7

0

0/7

0

0/7

0

0/7

0

1/7

14

7/7

100

72

0/7

0

0/7

0

0/7

0

1/7

14

2/7

29

7/7

100

96

0/7

0

0/7

0

1/7

14

1/7

14

4/7

57

7/7

100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
- Dissolved oxygen in all vessels: >= 70 % / >= 81 % (nominal value > 60 %) - Lethality in the control in the Definite-Test: 0 fish (nominal value 1 fish)