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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 October 2013 to 11 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

yes

Remarks:

Larger spacing factor, i.e. 5, was used for the test as the dose response was very shallow.

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

2008

Deviations:

yes

Remarks:

spacing factor for concnetrations was 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO International Standard 10706: “Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea)”, 2000-03-30.

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the control. In addition, the filter containing undissolved residue was kept for possible analysis.

Sampling:
Frequency: At the beginning and at the end of three intervals of 48 hours (nominal days 0 and 2, 4 and 6, 12 and 14, 18 and 20).
Volume: 2.0 ml
Storage: Samples not analysed on the day of sampling were stored in a freezer until analysis.

At the end of the refreshment period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 2.0 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

Preparation started with a loading rate of 5 mg/l applying a 30-minute treatment with ultrasonic waves followed by a 2-day period of magnetic stirring to ensure maximum dissolution in test medium. The obtained solutions containing undissolved material were filtered through a 0.45 µm membrane filter (Whatman, rc 55). Subsequently, the Water Soluble Fractions (WSFs) were used as the highest concentrations. Lower test concentrations were prepared by diluting the highest concentration in test medium. The final test solutions were all clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with a known history.

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

Validity of batch: 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.

Characteristics: To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

none

Hardness:

179-214 mg calcium carbonate per litre

Test temperature:

19-21 °C

pH:

7.8-9.0

Dissolved oxygen:

8.7-11

Salinity:

not measured

Nominal and measured concentrations:

Nominal concentrations:
The concentrations measured in freshly prepared WSFs at the beginning of each renewal period ranged between 926 and 1697 ng/l (average 1268 ± 328 (StDev) ng/l). Measured concentrations in the undiluted WSF were at a level of 10-39% of initial at the end of each refreshment period. The actual concentrations in the remaining groups showed a similar variability. Therefore, average exposure concentrations were calculated to correspond to 6.2, 6.8, 14, 83 and 550 ng/l in solutions containing 0.16, 0.8, 4.0, 20 and 100% of a WSF prepared at a loading rate of 5.0 mg/l, respectively.

Details on test conditions:

Test duration: 21 days
Test type: Semi-static
Frequency of renewal: Every 48 hours
Test vessels: Volume: 60 ml (6 x Ø 3.5 cm), all-glass covered with a Perspex plate.
Medium: M7
Experimental design: At the start of the experiment (nominal day 0) 10 neonate daphnids, less than one day old, per group were divided over ten vessels each containing a minimum of 50 ml test medium. The control group consisted of 20 daphnids.
Light: 16 h photoperiod daily;
: intensity at the start: 616-669 lux
: intensity at the end: 553-621lux
Feeding: Twice daily an amount of 0.25 ml of Chlorella pyrenoidosa suspension . On weekend days an amount of 0.50 ml was added in one single feed. This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211. From day 14 onwards, the total daily amount was increased to 0.75 ml, based on expertise. This daily ration corresponded to 0.3 mg C/Daphnia/day.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

6.8 ng/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

70 ng/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 1.3-150 ng/l

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

209 ng/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 69-698 ng/l

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

550 ng/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: -

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

83 ng/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: -

Details on results:

- Mortality of parent animals: 5% in the control and 30% in the average exposure concentrations of 14 and 550 ng/l. This mortality was not significantly different than in the control.
- No. of offspring produced per day per female: in the control 84 with %CV of 16. No reduction was observed at the two lowest concentrations. In the concentrations of 14 and 83 ng/l 14% reduction was observed and at the highest concentration 87% reduction of reproduction was observed.
- Body length of parent animals at the end of the test: 4.18, 4.16, 4.10, 4.15, 4.26 and 3.69 mm in 6.2, 6.8, 14, 83 and 550 ng/l, respectively
- Type and number of morphological abnormalities: none
- Type and number of behavioural abnormalities: none
- Time to first brood release: 10 days in the control and the four lowest concentrations, 19 in the highest concentration
- Other biological observations: Increasing number of immobilised juveniles with increasing concentration was observed.

Reported statistics and error estimates:

Following statistical procedures were used to determine the NOEC for reproduction and growth:

• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)
• Differences between treatments and the control: Williams Multiple Sequential t-test Procedure

Mortality of parental daphnids was analysed with Fisher’s Exact Binomial Test with Bonfferroni Correction.

EC-values for reproduction:
The EC10 and EC50 were determined using the Probit analysis using maximum likelihood regression with the probits of the percentages of cumulative reproduction at the end of the test as function of the logarithms of the corresponding concentrations.

All analyses were performed with ToxRat Professional 2.10.05 (ToxRat Solutions® GmbH, Germany).

The full reproduction test was preceeded with a range-finding test and two other full tests from which both were not valid. However, results obtained in these tests suggested that the dose-response is very shallow. Therefore, in the main test a higher spacing factor than required by the guideline was used.

Table 1 Nominal and mean measured exposure concentrations

Test substance^1 % WSFprep. at 5.0 mg/l	Mean exposure concentration (ng/l)				Average exposure concentration  (ng/l)
	Day 0-2	Day 6-8	Day 12-14	Day 18-20
0.16	7.6	5.0^2	5.0^2	7.1	6.2
0.80	5.0^2	9.8	5.0^2	7.3	6.8
4.0	13	12	14	15	14
20	122	82	62	64	83
100	459	615	706	418	550

1.      % WAF prepared at a loading rate of 5.0 mg/l

2.      Half of LOQ (LOQ=10ng/l)

Table 2         Group mean cumulative number of juveniles per surviving parent and reduction of reproduction at the end of the reproduction test

	4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline), concentration (ng/l)
	Control	6.2	6.8	14	83	550
Mean:	84	87	88	72	73	11
Std.Dev.:	13	10	17	15	15	6
n:	19	10	10	7	10	7
CV:	16	11	19	20	21	54
% Reduction:	n.a.	-3.0	-4.8	14*	14*	87*

n.a. – not applicable

*    Statistically significant (p<0.05)

Table 3         Mean cumulative number of immobilised offspring per parental daphnid and aborted eggs in the third reproduction test

Abnormality	4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline), concentration (ng/l)
	Control	6.2	6.8	14	83	550
Immobile offspring	6.0	7.7	3.0	22	25	43
Aborted eggs	n.o.	2	2	n.o	n.o	n.o

n.o. – not observed

Validity criteria fulfilled:

yes

Conclusions:

4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) did not affect reproduction of Daphnia magna at 6.8 ng/l after 21 days of exposure (NOEC).

Exposure to average exposure concentrations of 14 ng/l and higher induced significant inhibition of the reproductive capacity of the parental daphnids.

4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) caused not only reduction of reproduction but also delayed the onset of reproduction at the highest concentration tested.

Executive summary:

Daphnia magna, 21-day reproduction study with 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline).

 

The study procedures described in this report were based on the OECD guidelines for Testing of Chemicals: Guideline No. 211, 2012. In addition,the procedures were designed to meet the test methods and validity criteria of the ISO International Standard 10706, 2000, the Commission Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

 

The batch of 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) tested was a white to off-white powder with a purity of 97.3% and the substance was not completely soluble in test medium at the loading rates initially prepared.

 

A Water Soluble Fraction was prepared at a loading rate of 5.0 mg/l and used as the highest concentration. Lower test concentrations were prepared by diluting the highest concentration in test medium. The final test solutions were all clear and colourless.

 

The reproduction test was performed in a semi-static system, included 10 vessels per test concentration and 20 vessels for an untreated control group. Each of the vessels contained one neonate (<24h old) Daphnia magna in 50 ml test medium. Daphnids were exposed to solutions containing 0.16, 0.80, 4.0, 20 and 100% of the WSF prepared at 5.0 mg 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) per litre.

 

The study duration was 21 days and the test solutions were renewed every 48 hours. The daphnids were fed on a daily basis with a Chlorella pyrenoidosa suspension. Every workday the condition of the parental daphnids was recorded, during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test the lengths of the surviving parental daphnids were measured.

 

During the study samples for analyses were taken at the beginning and the end of four intervals of 48 hours.

 

Calculated average exposure concentrations corresponded to 6.2, 6.8, 14, 83 and 550ng/lin solutions containing 0.16, 0.80, 4.0, 20 and 100% of the WSF prepared at 5.0 mg/l, respectively.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

Mortality of parental daphnids observed in test concentrations was not statistically different from the control treatment.

 

The average cumulative number of young per female in the control after 21 days was 84. Significant reduction of reproduction was found at average exposure concentrations of 14 ng/l and higher. The onset of reproduction was clearly delayed in the highest group when compared to the control.

 

An increase of incidence of immobilised offspring was observed with increasing concentration.

 

Body length of adult daphnids was significantly reduced in the highest concentration.

 

4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) did not affect reproduction of Daphnia magna at 6.8 ng/l after 21 days of exposure (NOEC).

 

Exposure to average exposure concentrations of 14 ng/l induced significant inhibition of the reproductive capacity of the parental daphnids.

 

4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) caused not only reduction of reproduction but also delayed the onset of reproduction at the highest concentration tested.

Description of key information

NOECreproduction (21d) = 6.8 ng/L (measured) for the water flea Daphnia magna (OECD 211)
Adverse effects on reproduction occur at concentrations within the substances water solubility. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

6.8 ng/L

Additional information

A GLP-conform 21-day reproduction study is available for 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) (Tobor-Kaplon, 2014). Due to the low water solubility of the test item, a Water Soluble Fraction was prepared at a loading rate of 5.0 mg/L and used as the highest concentration. Lower test concentrations were prepared by diluting the highest concentration in test medium. The final test solutions were all clear and colourless. Daphnids were exposed to solutions containing 0.16, 0.80, 4.0, 20 and 100% of the WSF prepared at 5.0 mg 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) per litre. During the study samples for analyses were taken at the beginning and the end of four intervals of 48 hours. Calculated average exposure concentrations corresponded to 6.2, 6.8, 14, 83 and 550 ng/L in solutions containing 0.16, 0.80, 4.0, 20 and 100% of the WSF prepared at 5.0 mg/L, respectively. The average cumulative number of young per female in the control after 21 days was 84. Significant reduction of reproduction was found at average exposure concentrations of 14 ng/L and higher. The onset of reproduction was clearly delayed in the highest group when compared to the control. 4,4’-(9H-fluoren-9-ylidene)bis(2-chloroaniline) did not affect reproduction of Daphnia magna at 6.8 ng/L after 21 days of exposure (NOEC).