2,2'-(p-phenylenediethene-2,1-diyl)bisbenzonitrile

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 17 to November 1, 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Complete read across justification is attached in section 13. Source study has reliability 1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

part I, 1984

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling method
Actual test item concentrations were measured just before test start:
- duplicate samples from the single test medium (without daphnids)
- duplicate samples from the control (without daphnids)

after 48 hours (stability samples):
- duplicate samples from the single test medium (without daphnids)
- duplicate samples from the control (without daphnids)

For the 48-hour stability samples, additional flasks with adequate volumes of the freshly prepared test medium and the control were incubated during the test period under the same conditions as in the actual test (but without daphnids). Sampling from the test beakers with daphnids was not possible because the test medium volume (2 × 50 ml) was too small for the analytical requirements.
The concentration of the test item was analyzed in the duplicate test medium samples of the single test medium (undiluted filtrate) for both sampling times (0 and 48 hours). From the control samples, only one of the duplicate samples was analyzed from
each of both sampling times.
All samples were analyzed immediately after sampling since a reliable examination of the storage stability was not possible due to the very low solubility of the test item in test water.

Sample storage conditions before analysis: samples extracted with dichloromethane directly after sampling procedure.

Vehicle:

no

Details on test solutions:

The test medium was prepared as follows: due to the low water solubility of the test item, a supersaturated dispersion with a nominal concentration (loading rate) of 100mg/l was prepared by weighing 250 mg of the test item into 2500 ml test water. No auxiliary solvent or emulsifier was used. The test item was mixed into the test water as homogeneously as possible by ultrasonic treatment for 15 minutes and by intense stirring for 7 days at room temperature in the dark to dissolve a maximum concentration of the test item in the dispersion.
The long stirring period of the dispersion of 7 days was chosen according to the results of a pre-test (without GLP) which showed that the test item is hardly soluble in test water.
Then, the supersaturated stock dispersion of the test item was filtered through a glass microfibre filter (Whatman GF/C, maximum pore size approximately 1.2 µm) after the stirring period of 7 days. The undiluted filtrate of the supersaturated stock dispersion with the maximum concentration of dissolved and very finely dispersed test item was used as the only test medium. A filter with a pore size of approximately 1.2 µm was chosen since the test item concentrations in a filtrate filtered over a membrane filter with a smaller pore size of 0.45 µm were below the limit of quantification of the analytical method (pre-experiment without GLP).
The actual concentration of the test item in the test medium was analytically determined. The test medium was prepared just before introduction of daphnids (= start of the test). The test concentration was based on the results of a range-finding test and on the results of a pre-experiment to the solubility of the test item (without GLP). However, loading rates in excess of nominal 100 mg/l or test item concentrations above the maximum concentration which could be dissolved or very finely dispersed in test water were not tested according to the guidelines.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISMS
- Strain/clone: clone 5 of Daphnia magna Straus
- Source: University of Shiffield (UK)
- Age of test organisms: 6 - 24 hours old and not first brood progeny
- Feeding during test: none

ACCLIMATION
- Acclimation conditions (same as test or not): yes, regarding pH, main ions, total hardness, temperature and light conditions

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

2.5 mmol/l = 250 mg/l as CaCO3

Test temperature:

21 °C

Dissolved oxygen:

Test water was aerated until oxygen saturation. During test period, no aeration of test water.

Nominal and measured concentrations:

Analytically determined test item concentrations in test medium were 0.0093 and 0.011 mg/l at the start and the end of exposure period, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 2
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: less than 1 daphnia per 2 ml test solution

TEST MEDIUM / WATER PARAMETERS
- Alkalinity: 0.8 mmol/l
- Ca/Mg ratio: 4:1 (based on molarity)
- Na : K ratio: 10:1 (based on molarity)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light and 8-hour darkness with a 30-minute transition period (light intensity at light period between 300 and 800 Lux)

EFFECT PARAMETERS MEASURED: immobility or mortality after 24 and 48 h of exposure (those organisms not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile).

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: yes

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

or very finely dispersed

Basis for effect:

mobility

Details on results:

The analytically determined test item concentration in the samples from the freshly prepared test medium (the undiluted filtrate of the supersaturated dispersion) amounted to 0.0093 mg/l at the start of the test. In this test medium, incubated under the test conditions during the test period (but without daphnids), the measured concentration of test substance was 0.011 mg/l after 48 hours. Thus, test substance was stable under the test conditions over 48 hours. All biological results are related to the mean measured test item concentration of 0.010 mg/l (calculated as the average over all measurements in the undiluted filtrate).
In the control and at the single test item concentration of 0.010 mg/l, no immobilized or dead test organisms or other signs of intoxication were determined during the test period of 48 hours.

Therefore, the 48-hour NOEC and the 48-hour EC0 of test substance to Daphnia magna were determined to be at least 0.010 mg/l (loading rate of 100 mg/l). The 48-hour NOEC and the 48-hour EC0 might even be higher but concentrations of the test item above the maximum concentration which could be dissolved or very finely dispersed in the test water were not tested, according to the guidelines. The 48-hour EC50 and the 48-hour EC100 were clearly higher than the solubility limit of the test item in the test water. These values could not be quantified, since the test item had no toxic effect up to the highest concentration, which could be dissolved and very finely dispersed in the test water.
No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the whole test duration.

At the beginning and the end of the test period, the dissolved oxygen concentrations in the test medium and the control were at least 8.1 mg/l. The pH-values ranged from 7.8 to 8.0.
Duriing test period, test medium appeared as clear and no remarkable observations were done.

Effects on mobility

dilution	mean measured conc. (mg/l)	no. of daphnids tested	no. of immobilized daphnids		% of immobilized daphnids
			24 h	48 h	24 h	48 h
control	-	20	0	0	0	0
undiluted filtrate	0.010	20	0	0	0	0

Test item concentrations in test medium

hours	mg/l	average mg/l	average corrected by mean recovery of spiked samples (mg/l)
0	0.0058	0.0059	0.0093
0	0.0059
48	0.0066	0.0068	0.0106
48	0.0069
mean		0.0063	0.0101

Conclusions:

EC0 (48h) = 0.010 mg/l, i.e. the highest test item concentration, which can be dissolved or very finely dispersed in test water.

Executive summary:

Method

The acute toxicity of the test item to daphnia magna was determined in a 48-hour static test according to the EU method C.2 (1992) and the OECD guideline 202, part I (1984). A limit test was performed to demonstrate that the test item has no toxic effect on the test organisms up to and including the highest test item concentration which can be dissolved or very finely dispersed in test water. Due to the very low water solubility of the test item, a supersaturated dispersion of the test item with a loading rate of 100 mg/l was continuously stirred at room temperature in the dark over 7 days. Then, the dispersion was filtered. Only the undiluted filtrate with the maximum concentration of dissolved and very finely dispersed test item was used as the test medium. Additionally, a control was tested in parallel.

Results

The analytically determined test item concentration in the test medium were 0.0093 and 0.011 mg/l at the start and the end of the test period, respectively. Under the test conditions, the test item was stable during the test period of 48 hours. All biological results are related to the mean measured test item concentration of 0.010 mg/l, calculated as the average of all measurements.

In the control and in the undiluted filtrate, no immobilized or dead test organisms or other signs of intoxication were determined during the test period of 48 hours.

The 48-hour NOEC (highest concentration tested without toxic effects after the exposure period of 48 hours), and the 48-hour EC0 to daphnia magna were determined to be at least 0.010 mg/l (loading rate of 100 mg/l). The 48-hour NOEC and the 48-hour EC0 might even be higher but concentrations of the test item above the maximum concentration which could be dissolved or very finely dispersed in the test water were not tested, according to the guidelines. The 48-hour EC50 and the 48-hour EC100 were clearly higher than the solubility limit of test item in test water. These values could not be quantified, since the test item had no toxic effect on the daphnids at the highest test item concentration which could be dissolved or very finely dispersed in test water.

Description of key information

Highest concentration, which could be dissolved or very finely dispersed: 0.01 mg/l

EC0 (48h) = 0.010 mg/l

EC50 (48h) > 0.010 mg/l