Benzenamine, N-phenyl-, styrenated

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is highly insoluble in water, hence indicating that aquatic toxicity is unlikely to occur

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING

According to REACH Annex VI column 2, 9.1.4. Activated sludge respiration inhibition testing, the study does not need to be conducted if:
— there is no emission to a sewage treatment plant, or
— there are mitigating factors indicating that microbial toxicity is unlikely to occur, for instance the substance is highly insoluble in water, or
— the substance is found to be readily biodegradable and the applied test concentrations are in the range of concentrations that can be expected in the influent of a sewage treatment plant..

In general, there is no legal limit value available defining a substance as poorly water soluble or insoluble under REACH. However, in ECHA’s Guidance document Chapter R.7b: Endpoint specific guidance Version 3.0 – February 2016, it is stated: „poorly water soluble substances (e.g. water solubility below 1 mg/L or below the detection limit of the analytical method of the test substance)“. Further, poorly soluble substances are defined by OECD (2000 OECD SERIES ON TESTING AND ASSESSMENT, Number 23, GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES, ENV/JM/MONO(2000)6) as substances with a limit of solubility <100 mg/l although technical problems are more likely to occur at <1mg/l as defined in TGD (1996). Very low water solubility (i.e. in the low μg/l range) could be used as a reason to significantly modify a standard test or to test non-pelagic organisms preferentially.
ECHA’s Guidance document Chapter R.7c: Endpoint specific guidance Version 3.0 – June 2017 further says: „As indicated in the OECD TG 305, for strongly hydrophobic substances (log Kow > 5 and a water solubility below ~ 0.01-0.1 mg/L), testing via aqueous exposure may become increasingly difficult. However, an aqueous exposure test is preferred for substances that have a high log Kow but still appreciable water solubility with respect to the sensitivity of available analytical techniques, and for which the maintenance of the aqueous concentration as well as the analysis of these concentrations do not pose any constraints.
The solubility of the test item in water was determined to be 20.6 µg/l, determined based on the most soluble fraction. The additional components could not have been determined above their LOQ. Further, 9.44 µg/l was the LOQ of p-SDPA, making in case of a full and not limit test the determinations of the lower concentrations impossible. Last but not least, water solubility determination was performed in HPLC water. Ecotoxicity tests are performed in media containing various supplements and biological material, which is expected to lower the sensitivity of the analytical method. Hence, quantification of the test item up to its water solubility in ecotoxicity test media could like not be performed at all. So, the required analytics for ecotoxicity testing, both short and long-term, are technically not feasible, and testing does not need to be conducted.

Based on the available information it can be stated that, as outlined in the first waiving possibility under REACH, the substance is highly insoluble in water, and hence, aquatic toxicity is unlikely to occur. The latter conclusion is supported by available studies conducted prior to REACH implementation, i.e. each a study for toxicity against Brachydanio rerio, and studies according to EU method C.2 and OECD 209. In those studies, the EL50 was determined to be 0.92 mg/l (fish toxicity, WAF), EL50 ca. 50 mg/l (EU method C.2) and EC50 > 10 g/l (OECD 209), which are way above the possible water solubility of the registered substance. With regard to daphnids and fish, adequate information for environmental classification and labelling is available, as the noted effects were above the water solubility and trigger classification as Aquatic Chronic Cat. 4. So omitting this test would not result in an underestimation of the actual hazard, especially as Activated sludge respiration inhibition testing is not relevant for classification according to Regulation 1272/2008.

Hence, an additional study of the acute toxicity of the registered substance to algae does not need to be conducted, as it is considered both not technically feasible and no additional information on classification and labelling could be retrieved, and the study can be omitted.

Reason / purpose for cross-reference:

data waiving: supporting information

Remarks:

water solubility

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD 209 guideline study on the registered substance itself with minor deficiencies in documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: ETAD method No. 103 A screening test for the assessment of the possible inhibitory effect of a chemical substance on aerobic wastewater bacteria, 1979.

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

not specified

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct weighing
- Controls: no treatment / 3,5 dichlorophenol
- Evidence of undissolved material (e.g. precipitate, surface film, etc): product insoluble in water, adheres to glas surface

Test organisms (species):

activated sludge

Details on inoculum:

- Laboratory culture: Belebtschlamm Quelle Laborschlamm OECD-Anlage (activated sludge source laboratory sludge OECD facility)
- Initial biomass concentration: 0.30 g/l

Test type:

static

Water media type:

not specified

Limit test:

no

Test temperature:

20.6-22.6°C

pH:

5.9 - 8.3

Nominal and measured concentrations:

0, 313, 625, 1250, 2500, 5000, 10000 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Material, fill volume: glas, 250 ml
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1 (positive control) / 2 (negative control)
- No. of vessels per vehicle control (replicates): 2

EFFECT PARAMETERS MEASURED: respiration rate mg/l*min O2 / percentage inhibition

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 0, 313, 625, 1250, 2500, 5000, 10000 mg/l (nominal)

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Dose descriptor:

EC50

Effect conc.:

> 10 g/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: no information on duration given

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: product insoluble in water, adheres to glas surface

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 100% inhibition at 100 mg/l, 10.35% inhibition at 1 mg/l

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted according to OECD guideline 209 on the registered substance itself with minor deficiencies in documentation. However, the given data indicate that the study was well and scientifically reasonable performed. The positive control induced the appropriate response. Hence, the results can be considered as sufficiently reliable to assess the toxicity of the test item towards microorganisms. The determined EC50 was > 10000 mg/l, hence, the substance can be considered as non-toxic to activated sludge.

Executive summary:

The potential hazard of the test item towards microorganisms was tested in an OECD 209 guideline study. Concentrations ranging from 0 to 10000 mg/l were applied to activated sludge and the respiration rate was determined. Concentrations up to 10000 mg/l did not result in an inhibition of the respiration rate. 3,5-dichlorophenol served as positive control and induced the appropriate results. The test item was non-toxic to activated sludge, the EC50 was determined as > 10000 mg/l.

Description of key information

Conduction of the study technically not feasible.