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Short-term toxicity to fish

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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:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. certificate)
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 loadings 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 coal tar 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
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
Key result
Duration:
96 h
Dose descriptor:
LL0
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
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).

 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).
Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material ‘Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol (OAPP) [EC no. 700-960-7] consists of a complex mixture of numerous aryl-aromatic constituents some of them containing also a phenolic structural element. The constituents can basically be divided into groups corresponding to the presence of phenolic OH-groups as well as their level of oligomerisation (dimers, trimers).
The target substance ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised’ (NAF-AO) [EC no. 701-299-207] consists also of a complex mixture of numerous aryl-aromatic constituents but without phenolic substituents present. Its components can basically divided into groups corresponding to their level of oligomerisation (dimers, trimers, tetramers).
Both substances have various constituents in common (non-phenolic dimers, trimers and tetramers / higher oligomers). Their nature and their composition are considered to be sufficiently similar that short-term toxicity to fish will be affected in a similar way and resulting effects will be sufficiently similar. Therefore, the source substance OAPP is suited as supporting substance for NAF-AO with regard to short-term toxicity to fish, and data resulting from the source substance can be used for characterising the short-term toxicity to fish of the target substance NAF-AO.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source substance OAPP is a UVCP substance obtained in an acid catalysed oligomerisation / alkylation reaction of the starting materials 2-phenylpropene and phenol. In this process, aromatic and phenolic components react with each other resulting on the one hand in oligomerisation products of 2-phenylpropene (reaction of 2-phenylpropene molecules among themselves, aryl-aliphatic substances) and on the other hand in 2-phenylprop-2-yl substituted (methylstyrenated) phenols (reaction of 2-phenylpropene with phenol, aralkyl-substituted phenols, phenolic substances). Besides dimeric products also trimeric and oligomeric products can be formed. Aromatic rings within the products are connected by methyl-substituted alkenyl carbon-chains in case of the 2-phenylpropene oligomers and by a single bridging carbon atom substituted by two methyl groups in case of methylstyrenated phenols. Besides phenyl substituents, non-phenolic oligomers of OAPP can also include indan substituents.
Based on the degree of oligomerisation four to five basic groups of constituents are formed. Two or three groups contain purely aryl-aliphatic (non-phenolic) substances differing only in the degree of oligomerisation (dimers, trimers, and higher (n ≥ 4) oligomers). Two groups comprise the alkylation products of phenol (mono or di 2-phenylprop-2-yl (methylstyryl) substituted phenols; phenolic products). Non-phenolic constituents (aryl-aliphatic oligomers) amount together to about 45 to 80% of OAPP, while the phenolic components contribute about 20 to 50%.
OAPP is a viscous oily material with a low water solubility (between 1 and 3.5 mg/L depending on its composition).
Basically, there are two centres for metabolic transformation present in both component types. On the one hand, the aliphatic structure linking aromatic/phenolic rings and especially their methyl substituents can be metabolised by oxidative transformation finally leading to carboxylic acids and eventually to decarboxylation. The second major pathway for metabolism concerns the aromatic rings. Metabolism will result in hydroxylated species like phenols or quinones.
The target substance NAF-AO is a UVCB substance as well, also obtained in an acid catalysed alkylation and oligomerisation reaction of the starting material ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction’. This material comprises predominantly styrene and indene derivatives. In the production process, oligomerisation products of C8-10 aromatic hydrocarbons are formed consisting of dimeric, trimeric, and oligomeric (n ≥ 4) products. The individual constituents are the same as are produced in the production process of OAPP. Due to the composition of the starting material, no phenolic but only aryl-aliphatic products are formed. These products consist of two, three, or more aromatic rings connected by methyl-substituted alkenyl carbon-chains (dimers, trimers, oligomers). The aromatic substituents include besides benzene also indan. Based on the degree of oligomerisation, constituents are combined into three different groups (dimers, trimers and higher oligomers). Depending on the type of technical product (Novares L 100, L 700, TL 10), composition with respect to different component groups will vary (dimers from ca. 20 to 70%, trimers from ca. 10 to 30%, higher oligomers from ca. 15 to 50%).
NAF-AO is a viscous oily material with a low water solubility (between 0.6 and 1.3 mg/L depending on its composition). Thus, water solubility is somewhat lower than the water solubility of OAPP. Values for water solubility obtained by (Q)SAR (US EPA EPI suite) for individual model constituents are lower (between 0.082 and 0.038 mg/L for dimers and between 0.011 and 0.027 mg/L for trimers) indicating that measured values may overestimate the real water solubility. Overall, the water solubility of NAF-AO is low to very low depending on the technical product and its composition (higher percentages of trimers and higher oligomers will reduce the water solubility).
Basically, the metabolism of NAF-AO is likely to proceed in a similar way as in OAPP resulting in similar metabolic transformation products. Aliphatic structures with their methyl substituents linking aromatic rings can be metabolised by oxidative transformation finally leading to carboxylic acids and eventually to decarboxylation. The second sites for metabolic transformation are the aromatic rings present in the substance. Metabolism will result in hydroxylated species like phenols or quinones. Thus metabolism at aromatic rings can lead to similar products that are already present in the phenolic fraction of OAPP.

3. ANALOGUE APPROACH JUSTIFICATION
Environmental fate and ecotoxicological effects of complex mixtures like UVCB substances will be determined by their intrinsic properties. Under environmental conditions or during processing of the substances, environmentally available or volatile components can be released into the environment. Distribution properties and environmental toxicity will characterise its environmental behaviour.
The target substance NAF-AO and the source substance OAPP are very similar. They contain to a substantial extent the same constituents. They only differ in the presence of phenolic constituents, which are lacking in NAF-AO. But due to metabolic processes at the aromatic rings, phenolic compounds will be formed after uptake of NAF-AO.
Based on their molecular weight and structure, both substances can be absorbed and migrate through membranes. Metabolism will lead to similar products. Thus, toxic effects on fish are expected to be similar.
Due to the presence of phenolic constituents, the water solubility of OAPP is somewhat higher compared to NAF-AO. Phenolic constituents will directly be transported into the body and result in a higher body burden of reactive phenolic substances. Therefore, aquatic toxic effects of the source substance OAPP are likely to be more pronounced than that of the target substance NAF-AO. The presence of phenolic constituent is considered to increase the aquatic toxicity of OAPP thus being a worst case compared to NAF-AO.
For reasons summarised above, both substances are considered to be sufficiently similar that results of short-term fish toxicity tests of the source substance OAPP can be used to assess the short term toxic effects on fish caused by the target substance NAF-AO.
Reason / purpose:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol;
Reference: Aniol et al. 2010
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
Remarks on result:
other: the test result of the source substance is adopted for the target substance NAF-AO
Key result
Duration:
96 h
Dose descriptor:
LL0
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: the test result of the source substance is adopted for the target substance NAF-AO
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
behaviour
Remarks on result:
other: the test result of the source substance is adopted for the target substance NAF-AO

Description of key information

Data obtained for the substance OAPP (study according to OECD TG 414) are used to specify the short-term toxicity to fish of the substance NAF-AO in a read-across approach. The LL50 was determined to be 25.8 mg/L. Data are only available for freshwater.

Key value for chemical safety assessment

LC50 for freshwater fish:
25.8 mg/L

Additional information

For the substance NAF-AO, no data on the short-term toxicity to fish is available. Instead, data resulting from the supporting substance OAPP will be used. OAPP is produced in a similar process as NAF-AO producing overlapping constituents in both substances. The substances are considered to be sufficiently similar that data of the source substance OAPP on short-term toxicity to fish can also be used for the target substance NAF-AO in a read-across approach.

Short term toxicity to fish was investigated in a GLP compliant study according to OECD TG 203. Due to the complex composition of the test material and the variable water solubility of constituents, loadings of water accommodated fractions (WAF) were used as concentration values.

The LL50 (lethal loading) of OAPP was determined to be 25.8 mg/L. This value is adopted for the short-term toxicity to fish of NAF-AO.