Benzenamine, N-phenyl-, styrenated

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24.08. - 26.08.1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study on the registered substance itself, substance was applied as WAF which is a commonly accepted approach, as e.g. in ECHA's guidance R.7b.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

"Acute Toxicity for Daphnia" Council Directive 92/69/EEC C.2 (1992)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct weighing. To produce the WAF's, the test substance were weighed into water, treated for 60 seconds at 8000 rpm. with an ultra-turrax and afterwards stirred for 24 hours on a magnetic stirrer. To remove undissolved particles, the resulting emulsions were filtered using folded filters of the pore size 7-12 µm
- Controls: M4-Medium according to Elendt and BGA (1992)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): yes, see above

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain: Daphnia magna STRAUS, parthenogenetic females
- Source: laboratory bred at Bayer AG Leverkusen, origin: cloned at the Federal Health Office (BGA) in Berlin
- Age at study initiation (mean and range, SD): 0-24 h

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

14.7°dH

Test temperature:

20.3 - 20.5 °C

pH:

8.0 - 8.1

Dissolved oxygen:

8.4 - 8.5 mg/l (after 48h)

Nominal and measured concentrations:

nominal: 6.3, 12.5, 25, 50, 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: cylindrical test vessels
- Material, size, headspace, fill volume: diameter 4.0 cm, height 6.5 cm, 20 ml test medium
- Aeration: not ventilated
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4-Medium according to Elendt and BGA (1992)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light/8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- oxygen [mg/l]
- pH
- water temperature [°C]
- number of immobile daphnids after 24 hours and 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 6.3, 12.5, 25, 50, 100 mg/l (nominal, WAF)

Reference substance (positive control):

not required

Duration:

24 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

6.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

ca. 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Approximately 50 % effect was achieved at an EL of 50 mg/l

Details on results:

- Behavioural abnormalities: none stated
- Mortality of control: no mortalities occurred
- Other adverse effects control: none stated
- Abnormal responses: none stated
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: heterogeneous composition of the test substance, *Water Accommodated Fractions* (-WAF) of the test substance were tested. To produce the WAF's, the test substance were weighed into water, treated for 60 seconds at 8000 rpm. with an ultra-turrax and afterwards stirred for 24 hours on a magnetic stirrer. To remove undissolved particles, the resulting emulsions were filtered using folded filters of the pore size 7-12 µm.

Validity criteria fulfilled:

not specified

Conclusions:

The study was conducted under GLP according to EU method C.2 on the registered substance itself without analytical monitoring due to the substances intrinsic properties, i.e. poor water solubility. The present proceedings correspond to the preparation of a WAF, which is in line with the recommendations in ECHA's guidance R.7b. The method is hence to be considered scientifically reasonable. Hence, the results can be considered as reliable to assess the effects of the test item to aquatic organisms, here, daphnia magna. Taking into account the determined effect values after 48h, based on nominal concentrations, i.e. EL0 = 6.3 mg/l, EL100 > 100 mg/l, and approx. 50 % effect was achieved at an EL = 50 mg/l, based on motility, the substance does not need to be classified as aquatic acute cat. 1 according to Regulation (EC) No. 1272/2008.
According to Table 4.1.0 “Classification categories for hazardous to the aquatic environment” of the CLP regulation, Substances for which adequate chronic toxicity data are not available should be classified as Aquatic chronic Cat 3 if the “48 hr EC 50 (for crustacea) is > 10 to ≤ 100 mg/l … and the substance is not rapidly degradable and/or the experimentally determined BCF ≥ 500 (or, if absent, the log K ow ≥ 4)”. As the EL50 is approx. 50 mg/l, classification as such would be triggered. However, as the water solubility of the test item is 20.6 µg/l, this effects level clearly exceeds the water solubility, making classification as Aquatic chronic Cat 4 more appropriate.

Executive summary:

The 48hr acute toxicity of the test item to Daphnia magna Straus was studied under static conditions. Daphnids were exposed to control and test chemical at nominal concentrations of 6.3, 12.5, 25, 50, 100 mg/l for 48 hr, substance was tested as WAF’s. Immobilization was observed daily. The 48 – hour EL50 was approx. 50 mg/l based on immobilization.

Based on the results of this study, the test item would be classified as aquatic chronic cat. 4 to Daphnia magna in accordance with the classification system of Regulation (EC) No. 1272/2008.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Data waiving:

study technically not feasible

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 VII column 1, 9.1.1. Short-term toxicity testing on invertebrates (preferred species Daphnia) The registrant may consider long-term toxicity testing instead of short-term. According to column 2, 9.1.1. The study does not need to be conducted if:
— there are mitigating factors indicating that aquatic toxicity is unlikely to occur, for instance if the substance is highly insoluble in water or the substance is unlikely to cross biological membranes, or
— a long-term aquatic toxicity study on invertebrates is available, or
— adequate information for environmental classification and labelling is available.
The long-term aquatic toxicity study on Daphnia (Annex IX, section 9.1.5) shall be considered if the substance is poorly water soluble.

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 the third waiving criterion under REACH is also met.

Hence, an additional study of the acute toxicity of the registered substance against daphnids 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 due to animal welfare.

Reason / purpose for cross-reference:

data waiving: supporting information

Remarks:

water solubility

Reason / purpose for cross-reference:

data waiving: supporting information

Description of key information

Conduction of the study technically not feasible.