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Endpoint:
activated sludge respiration inhibition testing
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 209 (Activated Sludge, Respiration Inhibition 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:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water extract as Water Accommodated Fraction (WAF)
- Eluate: The WAF for the Activated Sludge, Respiration Inhibition Test as Limit Test was prepared by stirring an amount of 100 mg/L of the test item in tap water for 24 h with a magnetic stirrer.
The test loading rate of the test substance was weighed on a weighing scoop that afterwards was placed into the glass beaker. The beaker was then filled with tap water. The 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 WAF was allowed to stand for 1 h before use to facilitate phase separation. The extract gained with this method was clear.
- Differential loading: yes, only one loading tested
- Controls: yes, without test substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
activated sludge
Details on inoculum:
- Source: Activated sludge from a sewage treatment plant (Kläranlage Oberes Echaztal, Pfullingen)
- Pretreatment: The activated sludge was washed three times with tap water.
- Initial biomass concentration: suspended solids (dry weight) ~ 4 g/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
10 min for determination of respiration inhibition:
After 3 hours of aeration the contents of the test vessels were transferred into a measuring apparatus, and the respiration rate was measured and recorded over a period of up to 10 minutes.
Test temperature:
19.6 - 20.5 °C (20 ± 2 °C)
pH:
no data
Dissolved oxygen:
> 6.5 mg/L
Nominal and measured concentrations:
100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, fill volume: 2 L glass beaker, test volume 0.5 L
- Aeration: yes
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: dry weight of suspended solids 4 g/L (± 10 %)
- Conditioning: The test vessel was conditioned with the WAF for 0.5 h in order to saturate the potential adsorptive capacity of the glass walls. The spent medium was exchanged for fresh WAF-containing test medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water (blank controls) or WAF/tapwater (test) supplemented with "synthetic sludge feed" (cocktail of peptone, meat extract urea an minerals) in deionised water.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: Inhibition of O2-consumption (respiration)


Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
other: NOELR (No Observed Effect Loading Rate)
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: limit test (100 mg/L)
Key result
Duration:
3 h
Dose descriptor:
other: EL50 (Effect Loading at 50 % inhibition)
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: limit test (100 mg/L)
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 14.4 mg/L

The EC50 (EC = Effective Concentration) of the Reference item 3,5-dichlorophenol was determined byusing Probit-analysis (Finney-method, log-normal distribution; confidence limit 95 %, significance level: 0.05)
Concentr. Respiration Inhibition (%)
(mg/L) [mg O2/(L x h)
==============================
3 61.5 11.3
10 45.7 34.1
30 18.0 74.1
==============================
Reported statistics and error estimates:
not applicable

Results of the Activated Sludge, Respiration Inhibition Test of Novares LA 300

Loading rate (mg/L)

Respiration rate

[mg O2/(L x h)]

Inhibition (%)

Control 1

-

64.9

-

Control 2

-

73.9

-

Test item

100

64.9

6.5*

Remark: * With reference to the validity criterion of OECD-Guideline 209 that recommends a deviation of respiration rates of the two controls of ≤15 %, the result of inhibition of theTest Item of 6.5 % was not regarded as a significant inhibition.

Validity criteria fulfilled:
yes
Remarks:
- Deviation between respiration rates of the controls: 12.2% (nominal value ≤ 15%); - EC50 (3 h) of 3,5-dichlorophenol: 14.4 mg/L (nominal value 5 - 30 mg/L); - Dissolved oxygen in all vessels: ≥ 6.5 mg/L (nominal value ≥ 3 mg/L).
Conclusions:
Water Accommodated Fractions (WAFs) of Novares LA 300 (Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol, OAPP) caused no acute toxic effects on bacteria in an Activated Sludge, Respiration Inhibition Test according to OECD-Guideline 209 (carried out as Limit Test). A 3h EL50 of > 100 mg/L and a NOELR of 100 mg/L have been determined.
Endpoint:
activated sludge respiration inhibition testing
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 effects on aqueous microorganisms are equivalent. Therefore, the source substance OAPP is suited as supporting substance for NAF-AO regarding toxic effects to aqueous microorganisms, and data resulting from the source substance can be used for characterising the toxicity to aqueous microorganisms 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 the same 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
Under environmental conditions or in an aquatic environment, the source substance OAPP and the target substance NAF-AO will be released according to their physico-chemical properties and their ability to dissolve in water. Due to the presence of phenolic components, aqueous solubility of OAPP will be somewhat higher than the solubility of NAF-AO. Physico-chemical properties of both substances are considered to be sufficiently similar that uptake by microorganisms is assumed to be comparable. Due to the presence of phenolic constituents in OAPP, its toxic effects are considered to be more pronounced than toxic effects of NAF-AO. Therefore, results from toxicity test on aquatic microorganisms with OAPP as test material are considered a worst case compared to the substance NAF-AO.
For these reasons, both substances are considered to be sufficiently similar that results of toxicity tests on aquatic microorganisms with the source substance OAPP as test material can be used to assess toxic effects on aquatic microorganisms 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:
3 h
Dose descriptor:
other: NOELR (No Observed Effect Loading Rate)
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: limit test (100 mg/L)
Remarks:
the test result of the source substance is adopted for the target substance NAF-AO
Key result
Duration:
3 h
Dose descriptor:
other: EL50 (Effect Loading at 50 % inhibition)
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: limit test (100 mg/L)
Remarks:
the test result of the source substance is adopted for the target substance NAF-AO
Conclusions:
The study was carried out with the supporting substance (source substance) 'Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol' [EC no 700-960-7) (OAPP). Water Accommodated Fractions (WAFs) of the test item (Novares LA 300) caused no acute toxic effects on bacteria in an Activated Sludge, Respiration Inhibition Test according to OECD-Guideline 209 (carried out as Limit Test). A 3h NOELR of 100 mg/L and 3h EL50 of > 100 mg/L have been determined.
The test results of the source substance OAPP are adopted for the target substance NAF-AO.

Description of key information

In an Activated sludge respiration inhibition test with the supporting substance ‘Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol’ (OAPP), a 3-h NOELR (No Observed Effect Loading Rate) of 100 mg/L and a 3-h EL50 (Effect Loading causing 50 % inhibition) was observed.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

No data on the toxicity to aquatic microorganisms is available for the substance ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised’ (NAF-AO). Instead, results obtained with the structure related substance ‘Oligomerisation and alkylation reaction products of 2-phenylpropene and phenol’ (OAPP) are used (read across).

Both substances are UVCB with an overlapping range of constituents. Water solubility is low and not clearly defined due to the differing solubility of individual constituents. Depending on the quantity used, the concentration of the material dissolved in saturated water samples is variable. Therefore, water accommodated fractions (WAFs) have been used in aquatic toxicity tests.

The toxicity of the source substance OAPP to aquatic microorganisms has been determined in a study (limit test) according to OECD TG 209 under GLP conditions. A NOELR of 100 mg/L and an EL50 of > 100 mg/L was determined after exposure of activated sludge of a predominantly domestic sewage for a three hour period.

Based on the similarity of both substances, the test result of the source substance OAPP is adopted for the target substance NAF-AO.