Phenol, mono- + distyrenated (MSP+DSP)

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:

other: GLP 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 LS 500; phenol, styrenated
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, batch No. 28324
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LS 500_phenol, styrenated
- Substance type: organic
- Purity test date: 30.11.2012
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable

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) 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.
The beakers were equipped with a glass tube that allowed the separation of the water phase by siphoning.
The test loading rates of LS 500 weighed on watch-glasses, 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, 1.0, 2.5, 5.0, 10, and 25 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
- 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%

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:

23 +-2 °C

pH:

8.2 - 8.5

Dissolved oxygen:

87 - 99 % of saturation

Salinity:

--

Nominal and measured concentrations:

nominal: 1.0, 2.5, 5.0, 10, and 25 mg/L
measured (as TOC):
Period Control 1.0 mg/L 5.0 mg/L 25 mg/L (loading)
===================================================
0-24 h start 1.4 2.5 2.9 2.6 mg/L (TOC of WAF)
0-24 h end 1.5 2.8 3.0 2.6 mg/L (TOC of WAF)
72-96 h start 1.6 2.9 3.1 3.9 mg/L (TOC of WAF)
72-96 h end 1.8 3.0 3.1 3.9 mg/L (TOC of WAF)
===================================================
Initial TS-related TOC values were about 1 mg/L (at 1.0 mg TS/L), about 1.5 mg/L (at 5.0 mg TS/L),
and 1.5 – 2.5 mg/L (at 25 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:

LC50

Effect conc.:

1.77 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks on result:

other: value was derived from loadings by taking into account the corresponding measured TOC values at different time points and relating these values to the carbon content of the substance (see below).

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

14.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

1 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.

- 1.0 mg/L: Comparable to control during all observation periods.

- 2.5 mg/L: Activity was slightly reduced during the observation periods of 0 – 24 h, 24 – 48 h,
48 – 72 h and 72 – 96 h, surfacing could be observed at the observation period of 48 – 72 h.

- 5.0 mg/L: Activity was 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 and surfacing could also be observed.

- 10 mg/L: Activity was slightly reduced during the observation period of 0 – 24 h, and the fish partially could be found
at the bottom of the test vessels. Activity was definitely reduced at the observation periods of 24 – 48 h,
48 – 72 h and 72 – 96 h. The fish partially could be found at the bottom of the test vessels but also showed
agitated swimming movements, surfacing and balance disorder. Two fish died during the observation period
of 24 – 48 h, one more fish died during the observation period of 48 – 72 h.

- 25 mg/L: Activity was definitely reduced during the observation periods of 0 – 24 h, 24 – 48 h, 48 – 72 h and 72 – 96 h.
The fish could be found mainly at the bottom of the test vessels but also showed agitated swimming movements
and balance disorder. Three fish died during the observation period of 48 – 72 h, two more fish died during
the observation period of 72 – 96 h.


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

- Control: All fish fed immediately.
- 1.0 to 5 mg/L: All fish fed immediately.
- 10 mg/L: The surviving four fish fed with retardation compared to the control.
- 25 mg/L: The surviving two fish did not feed.

After the end of the Definite Test the fish were kept in de-chlorinated tap water from 13.02.10 to 16.02.10. During this period,
fish derived from the test loading rates of 1.0, 2.5, 5.0 and 10 mg/L recovered and showed normal behaviour.
The fish derived from the test loading rate of 25 mg/L slightly recovered and fed, but still with retardation, compared to the control
and the fish derived from the test loading rates of 1.0, 2.5, 5.0 and 10 mg/L.

Sublethal observations / clinical signs:

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

Time [h]

Control

1.0 mg/L

2.5 mg/L

5.0 mg/L

10 mg/L

25 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

0/7

0

2/7

28.6

0/7

0

72

0/7

0

0/7

0

0/7

0

0/7

0

3/7

42.8

3/7

42.8

96

0/7

0

0/7

0

0/7

0

0/7

0

3/7

42.8

5/7

71.4

Cumulative lethality after 96 h was 0 % at the test loading rates of 1.0, 2.5 and 5.0 mg/L.

Cumulative lethality after 96 h was 42.8 % at the test loading rate of 10 mg/L and 71.4 % at the test loading rate  of 25 mg/L.

Due to restricted solubility and multi-constituent composition of the test substance, the test was performed using water accommodated fractions. A 96h LL50 of 14.8 mg/L was determined (nominal load).

Dissolved concentrations of controls and selected test solutions were measured at the beginning and at the end of day 1 and day 4. TOC was analysed as representative parameter for the test substance. As nominal loads differ substantially from measured concentrations, the substance concentration representing 50% lethality is calculated from the nominal loads. From daily geometric means, geometric means for the test period were computed. After subtraction of the control value, mean measured concentrations were obtained.

 

	Loadings (mg substance/L)
	control	1 mg/L	5 mg/L	25 mg/L
	measured concentrations (mg TOC/L)
Time (h)
0	1.4	2.5	2.9	2.6
24	1.5	2.8	3.0	2.6
72	1.6	2.9	3.1	3.9
96	1.8	3.0	3.1	3.9
geometric mean	1.57	2.79	3.02	3.18
corrected by control		1.22	1.45	1.61

 

From the blank corrected, measured concentrations, a LC50 was derived from LL50 by correlating the measured concentrations to the nominal loads. Conversion to test substance concentrations was performed by applying a conversion factor of 1.163 resulting from mean carbon content of the test substance (86.018 %) determined by carbon content and percentage of the individual components of phenol, styrenated.

 

Loading (mg substance/L)	Concentration (mg TOC/L)	Concentration (mg substance/L)
96h LL50	96h LC50	96h LC50
14.8	1.52	1.77

 

Description of key information

Short-term toxicity of phenol, styrenated to fish was examined in two studies according to OECD TG 203. Both are considered to be valid. The lower of the two result will be used for further consideration.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.67 mg/L

Additional information

For the substance phenol, styrenated, two short-term toxicity studies to fish have been identified. One is a GLP conform study according to OECD TG 203 using Novares LS 500 as test substance (Aniol/STZ 2010). Reliability is 1. The study is used as key study.

The second study was carried out for the National Institute of Technology and Evaluation/Ministry of the Environment of Japan and is reported in the Japanese databases CHRIP/J-CHECK (CHRIP/NITE 1997). The study was performed according to OECD TG 203, but reporting is very limited. Details on test performance and results are not available from the database. The substance tested was phenol, styrenated (CAS 61788-44-1). Composition of the test substance is not reported, but from the substance name of the regulatory program (mono (or di or tri)-(alpha-methylbenzyl) phenol), it can be concluded that the test substance is a mixture of mono-, di-, and tristyrenated phenol. Similarity with Novares LS 500 is considered to be adequate to use data from this test as supporting evidence for the characterisation of Novares LS 500.

Aniol/STZ 2010

Due to restricted solubility and multi-constituent composition of the test substance, the test was performed using water-accommodated fractions. A 96h LL50 of 14.8 mg/L was determined (nominal loading).

Dissolved concentrations of controls and selected test solutions were measured at the beginning and at the end of day 1 and day 4. TOC was analysed as representative parameter for the test substance. As nominal loadings differ substantially from measured concentrations, the substance concentration representing 50% lethality is calculated from the nominal loadings. From daily geometric means, geometric means for the test period were computed. After subtraction of the control value, mean measured concentrations were obtained.

 

	Loadings (mg substance/L)
	control	1 mg/L	5 mg/L	25 mg/L
	measured concentrations (mg TOC/L)
Time (h)
0	1.4	2.5	2.9	2.6
24	1.5	2.8	3.0	2.6
72	1.6	2.9	3.1	3.9
96	1.8	3.0	3.1	3.9
geometric mean	1.57	2.79	3.02	3.18
corrected by control		1.22	1.45	1.61

 

From the blank corrected, measured concentrations, a LC50 was derived from LL50 by correlating the measured concentrations to the nominal loadings. Conversion to test substance concentrations was performed by applying a conversion factor of 1.163 resulting from mean carbon content of the test substance (86.018 %) determined by carbon content and percentage of the individual components of phenol, styrenated.

 

Loading (mg substance/L)	Concentration (mg TOC/L)	Concentration (mg substance/L)
96h LL50	96h LC50	96h LC50
14.8	1.52	1.77

 

As supporting evidence, read-across from the substance LA 300 (phenol, methylstyrenated) was used. Transformation of loadings to measured concentrations results in a 96h LC50 in the same order of magnitude as for LS 500 (phenol, styrenated).

CHRIP/NITE 1997

The test was performed according to OECD TG 203. Test conditions are not reported. It may be that a solubilising agent was used. This could explain the slightly higher LC50 determined in this test.

96h LC50:         5.6 mg/L.

 

The lower one of the two LC50 values (1.67 m g/L) is taken for further consideration.