Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol

Short-term toxicity to fish

Link to relevant study record(s)

Reference

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Novares LA 300 (phenol, methylstyrenated)
- Lot/batch No.: 28166
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LA 300_phenol, methylstyrenated
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable.
- Storage condition of test material: room temperature, exclusion of light

Analytical monitoring:

yes

Details on sampling:

Samples of the control and various test loading rates of the Definite Test were taken for TOC analysis (TOC=Total Organic Carbon) from separate vessels without fish at the start and end of test periods 0 - 24 h and 72 - 96 h of the Definite Test to assess the stability of exposure concentrations.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fractions (WAFs) were prepared by stirring various amounts of the test item in dechlorinated tap water
for 24 h with magnetic stirrers. The WAFs were prepared in 2-L glass beakers for the Range-Finding Test and
in 20-L solid-glass fish tanks for the Definite Test.
The beakers were equipped with a glass tube that allowed the separation of the water phase by siphoning.
The test loading rates of the test item were weighed on weighing scoops that afterwards were placed into the glass vessels.
The glass tanks 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, 5 mg/L, 10 mg/L, 25 mg/L, 50 mg/L, 100 mg/L
- 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 (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- 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%

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Total hardness 8.97 °dH
Carbonate hardness 6.98 °dH

Test temperature:

21.1 - 21.8 °C (23 +-2 °C)

pH:

8.2 - 8.5

Dissolved oxygen:

91 - 98 % of saturation

Salinity:

--

Nominal and measured concentrations:

nominal: 5.0, 10, 25, 50, and 100 mg/L
measured (as TOC):
Period Control 5 mg/L 25 mg/L 100 mg/L (loading)
===================================================
0-24 h start 1.0 2.5 3.1 3.7 mg/L (TOC of WAF)
0-24 h end 1.3 2.6 4.0 3.5 mg/L (TOC of WAF)
72-96 h start 1.4 3.2 4.6 -- mg/L (TOC of WAF)
72-96 h end 1.6 3.3 4.9 -- mg/L (TOC of WAF)
===================================================
Initial TS-related TOC values were about 1.5 mg C/L (at 5 mg TS/L), about 2.5 mg C/L (at 25 mg TS/L),
and ~2.5-3.0 mg/L (at 100 mg TS/L).

Details on test conditions:

TEST SYSTEM
- 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 based on previous pre-test using 1, 10, and 100 mg/L with 2 fish each

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

25.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Details on results:

Observations:

- Control: Swarming behaviour and activity were normal during all observation periods.

- 5 mg/L: Swarming behaviour and activity were comparable to control at the observation periods of 0 – 24 h, 48 – 72 h and 72 – 96 h.
Activity was slightly reduced at the start of the observation period of 24 – 48 h.

- 10 mg/L: Activity was slightly reduced during the observation periods of 0 – 24 h, 24 – 48 h, 48 – 72 h and 72 – 96 h,
the fish partially could be found at the bottom of the test vessels.

- 25 mg/L: Activity was definitely reduced during the observation periods of 0 – 24 h and 24 – 48 h, the fish partially could be found
at the bottom of the test vessels, and at the end of the observation period of 24 – 48 h, two fish had died.
The surviving fish showed balance disorder, agitated swimming movements, and one fish was apathetic.
At the end of the observation period of 48 – 72 h, two more fish had died, the surviving fish still showed balance disorder,
agitated swimming movements and, one fish was apathetic. At the end of the observation period of 72 – 96 h,
one more fish had died, and the surviving two fish could be found at the bottom of the test vessels, showing balance disorder.

- 50 mg/L: Activity was definitely reduced during the observation periods of 0 – 24 h and 24 – 48 h, the fish partially could be found
at the bottom of the test vessels but also showed balance disorder, agitated swimming movements, and one fish was apathetic.
At the end of the observation period of 48 – 72 h, two fish had died, the surviving fish could be found at the bottom of
the test vessels but also showed balance disorder, agitated swimming movements, and one fish was apathetic.
At the end of the observation period of 72 – 96 h, three more fish had died, and the surviving two fish could be found
at the bottom of the test vessels, showing balance disorder and also agitated swimming movements.

- 100 mg/L: Activity was definitely reduced during the observation period of 0 – 24 h, and all fish could be found at the bottom of
the test vessels, showing balance disorder. At the end of the observation period of 24 – 48 h, two fish had died,
the surviving fish could be found at the bottom of the test vessels, showing strong balance disorder, and one fish was apathetic.
At the end of the observation period of 48 – 72 h, the remaining five fish had died.



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

- Control: All fish fed immediately.
- 5 mg/L: All fish fed immediately.
- 10 mg/L: Five fish fed immediately, two fish fed with retardation compared to the control.
- 25 mg/L: The surviving two fish did not feed.
- 50 mg/L: One of the surviving two fish fed with retardation compared to the control, one fish did not feed.
- 100 mg/L: -


After the end of the Definite Test the fish were kept in de-chlorinated tap water from 06.02.10 to 08.02.10.
During this period, fish derived from the test loading rates of 5 and 10 mg/L recovered and showed normal behaviour.
The fish derived from the test loading rate of 25 mg/L slightly recovered, and one fish fed immediately at the end of the period on 08.02.10,
the other fed with retardation.

Reported statistics and error estimates:

The LL50 (LL=Lethal Loading) of the lethality in the Fish Test was determined by using Probit-analysis (Finney-method, lognormal distribution;
confidence limit 95 %, significance level: 0.05).

Sublethal observations / clinical signs:

 Number of dead fish and cumulative lethality [%] at various observation times (Report, Table 6):

Time [h]

Control

5 mg/L

10 mg/L

25 mg/L

50 mg/L

100 mg/L

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Number

[%]

24

0/7

0

0/7

0

0/7

0

0/7

0

0/7

0

0/7

0

48

0/7

0

0/7

0

0/7

0

2/7

28.6

0/7

0

2/7

28.6

72

0/7

0

0/7

0

0/7

0

4/7

57.1

2/7

28.6

7/7

100

96

0/7

0

0/7

0

0/7

0

5/7

71.4

5/7

71.4

7/7

100

Cumulative lethality after 96 h was 0 % at the test loading rates of 5 and 10 mg/L. Cumulative lethality after 96 h was 71.4 % at the test loading rates of 25 and 50 mg/L and 100 % at the test loading rate of 100 mg/L.

Results from dose-range finding (Report, Table 3):

Time [h]	Control		1 mg/L		10 mg/L		100 mg/L
	Number	[%]	Number	[%]	Number	[%]	Number	[%]
24	0/2	0	0/2	0	0/2	0	2/2	100
48	0/2	0	0/2	0	0/2	0	2/2	100

Validity criteria fulfilled:

yes

Remarks:

1) Dissolved oxygen in all vessels: >= 85 % / >= 91 % (nominal value > 60 %); 2) Lethality in the control in the Fish Tests: 0 fish / 0 fish (nominal value ≤ 1 fish in the Definite Test).

Description of key information

Short-term toxicity of 'Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol' (OAPP) to fish was examined in a valid GLP guideline study (OECD TG 203). Due to intrinsic properties of the test substance (UVCB substance), short-term fish toxicity was determined based on loadings. The effective loading resulting in 50 % mortality (EL50) was identified to be 25.8 mg/L test substance.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

25.8 mg/L

Additional information

For OAPP as UVCB substance, solubility can be variable at different substance concentrations. Therefore, effects observed are based on loadings/loading rates (nominal values). The EL50 for short-term toxicity to fish was determined to be 25.8 mg/L (effective loading).