4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-29 - 2017-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also conducted according to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Principles of method if other than guideline:

In addition, the following guidance document was followed:
Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

2015-11-03

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: 1. At the start of the test and after 24 hours from the freshly prepared solutions before distribution over the replicate vessels; 2. At the first renewal (t=24h) and the end of the test from the 24-hour old solutions after pooling solutions.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Saturated solutions (SS) of the test item, being insoluble into water (water solubility < 52 μg/L), were individually prepared at two loading rates of 5.0 and 100 mg/L. A 29-minute period of ultrasonic waves preceded a two-day magnetic stirring period in order to ensure reaching maximum dissolution in test medium. The obtained dispersions were filtered through a 0.45 μm membrane filter (RC55, Whatman) to remove the non-dissolved test item and used as test concentrations. Both solutions were clear and colorless at the end of the preparation procedure. Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Age at study initiation (mean and range, SD): < 24 hours
- Method of breeding: Cyclical parthenogenesis; with daphnids less than 3 days old, by placing about 250 of them in 5L of medium; cultures maximum 4 weeks old; fed a suspension of fresh water algae daily.
- Source: In-house laboratory culture
- Age of parental stock (mean and range, SD): > 2 weeks old
- Feeding during test : no feeding

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 - 21 °C

pH:

8.0 - 8.2

Dissolved oxygen:

8.2 - 8.8 mg/L

Nominal and measured concentrations:

Nominal: control, 5 and 100 mg/L (loading rates)
Measured concentrations: 0, 1.3 and 1.7 µg/L (average exposure concentration - 4 measurements)

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Volume of solution: 50 mL
- Aeration: No aeration
- Renewal rate of test solution: renewal of test solutions after 24h
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse Osmosis water
- Medium M7

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility after 24 and 48h

RANGE-FINDING STUDY
- Test concentrations: range-finding from 2014 chronic study (see Long-term toxicity to aquatic invertebrates)
- Results used to determine the conditions for the definitive study: No mortality/immobility over a 7-day period

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.7 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Analysis of the samples taken from the freshly prepared solutions showed measured concentrations between 0.9 and 2.2 μg/L for the loading rate of 5.0 mg/L and between 1.3 and 2.7 μg/L for the loading rate of 100 mg/L. These concentrations decreased by 10 to 41% and by 17 to 37% below initial during the 24-hour test periods for the loading rates of 5.0 and 100 mg/L, respectively. Based on these results, the average exposure concentrations were calculated.

Results with reference substance (positive control):

The 48h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.66 mg/L.
The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L.

Incidence of Immobility in the Final Test

Test group 4,4’-(9H-fluoren-9- ylidene)bis(2-chloroaniline) Loading rate (mg/L) / Average meas. (μg/L)	Vessel Number	Number Daphnia Exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
5.0 / 1.3	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
100 / 1.7	A B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) exceeded an average measured concentration of 1.7 μg/L in a loading rate that was prepared at 100 mg/L and thus exceeded the maximum soluble concentration that could be obtained in test medium under the test conditions.

Executive summary:

 A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 202 and also the Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000 under GLP conditions.

 

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at two nominal loading rates of 5 and 100 mg/L for 48 hours at a temperature of ca. 21 °C under semi-static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

The 48-Hour EL50 for the test item to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF, equivalent to greater than 1.7 µg/L based on measured concentrations (EC50). This indicates that, in the range of its water solubility, the test item was not toxic under the test conditions.

Description of key information

EC50 (48h) > 1.7 µg/L (measured) for Daphnia magna (OECD 202).

No toxicity observed at the limit of solubility of the test substance.

A second study from 1991 is available. However, that second study used a solubilizer to increase the concentration of the test substance in the medium. The observed effects on the mobility of Daphnia magna are observed at concentrations exceeding the solubility of the test material (EC50 1.1 mg/L for a solubility < 0.052 mg/L). Therefore, this result was considered as irrelevant.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

One reliable study is available on the short-term toxicity of 4,4´-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (CAS No. 107934-68-9) to aquatic invertebrates. The study was performed according to OECD guideline 202 and GLP, with water fleas (Daphnia magna) as test organisms. The daphnids were exposed to the substance for 48 hours at measured test concentrations of 1.3 and 1.7 µg/l (equivalent to nominal concentrations of 5 and 100 mg/L). Up to the measured concentration of 1.7 µg/l no effects on the mobility of the test organisms occurred.