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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

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

Adequacy of study:

key study

Study period:

From October 16 to November 2, 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Semi-static test with a test medium renewal every day, duplicate samples were taken from the freshly prepared test medium of the single test item concentration (undiluted filtrate) and the control at the start of the test (day 0) and at the last test media preparation on day 3.
To confirm the maintenance of the test item concentration during the test medium renewal periods of one day, duplicate samples were taken out of the test medium of the single test item concentration and the control at the end of the first test medium renewal period and at the end of the last test medium renewal.
All samples were taken from the approximate center of the aquaria without mixing of the test medium.
The concentrations of the test item were analyzed in all test medium samples from the single test item concentration (undiluted filtrate). From the control samples, only one of the duplicate samples was analyzed from the corresponding 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.

Vehicle:

no

Details on test solutions:

The test medium was prepared at each preparation date as follows: due to the low water solubility of the test item, a supersaturated dispersion with a nominal concentration (loading rate) of 100 mg/l was prepared by weighing 500 mg of the test item into 5000 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 glass microfibre filter (Whatman GF/C, maximum pore size approximately 1.2 µm) after the 7 days stirring period just before each preparation of the test medium. 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 test medium was prepared just before introduction of the fish (= start of the test) and prior to each test medium renewal.
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):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: Zoohaus Schaub, CH-4410 Liestal, Switzerland
- Age at study initiation (mean and range, SD):
- Length at study initiation: 3.0 ± 0.1 cm
- Weight at study initiation: 0.23 ± 0.05 g

ACCLIMATION
- Acclimation period: one week
- Acclimation conditions: same as test as for water and temperature
- Type and amount of food during acclimation: commercial fish diet
- Feeding frequency during acclimation:until one day before test
- Health during acclimation (any mortality observed): no detah, all fish were healthy

FEEDING DURING TEST: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

2.5 mmol = 250 mg/l as CaCO3

Test temperature:

21 - 22 °C

Dissolved oxygen:

Test water aerated until oxygen saturation.

Nominal and measured concentrations:

Based on a mean recovery of 63 %, average concentrations found in the treatment samples were corrected, resulting in 0.0039 and 0.0029 mg/l (non-aged samples) and 0.0035 and 0.0018 mg/l (aged samples).
The losses in the aged samples could be due adsorption, resulting from the low solubility of the test item in test water. .

Details on test conditions:

TEST SYSTEM
- Test vessel: 4
- Aeration: slightly aerated
- Renewal rate of test solution (frequency/flow rate): every day
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1g fish wet weight / l test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Alkalinity: 0.8 mmol/l
- Ca/Mg ratio: 4:1 based on molarity
- Na/K ratio: 10:1 based on molarity
- CaCl2 × 2H2O: 2 mmol = 294 mg/l
- MgSO4 × 7H2O: 0.5 mmol/l = 123 mg/l
- NaHCO3: 0.075 mmol/l = 65 mg/l
- KCl = 0.075 mmol/l = 5.8 mg/l

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light to 8-hour darkness photoperiod, with a 30 minute transition period (light intensity at light period approximately 50 to 500 Lux)

EFFECT PARAMETERS MEASURED: fish observed for symptoms of intoxication and mortality after 3, 24, 48, 72 and 96 h

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.003 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

and very finely dispersed

Basis for effect:

mortality (fish)

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 0.0039 and 0.0029 mg/l at the start of the test. After the test medium renewal period of one day, the measured concentrations slightly decreased to 0.0035 and 0.0018 mg/l. This slight decrease of the measured test item concentrations might have been caused by a precipitation of the finely dispersed test item, or due to a loss by adsorption onto the glass surfaces. All biological results are related to the mean measured test item concentration of 0.0030 mg/l (calculated as the average over all measurements in the undiluted filtrate during the test period).

In the control and in the undiluted filtrate with a mean measured test item concentration of 0.0030 mg/l, no mortality or other signs of intoxication were determined at the test fish during the test period of 96 hours.

Therefore, the 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours), and the 96-hour LC0 to zebra fish were determined to be at least 0.0030 mg/l (loading rate of 100 mg/l). The 96-hour NOEC and the 96-hour LC0 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 96-hour LOEC (lowest concentration with toxic
effects), the 96-hour LC50 and the 96-hour LC100 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 or 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.
The pH values in the test medium and the control ranged from 7.8 to 8.0. The oxygen concentration was always 7.9 mg/l or higher, and thus higher than 60 % oxygen saturation. The water temperature ranged from 21 to 22 °C.

Sublethal observations / clinical signs:

Concentration in test medium

day	age of sample	mg/l	average mg/l	average corrected by mean recovery of spiked samples in mg/l
0	0	0.0026	0.0025	0.0039
	0	0.0023
1	24	0.0020	0.0022	0.0035
	24	0.0024
3	0	0.0020	0.0019	0.0029
	0	0.0017
4	24	0.0011	0.0012	0.0018
	24	0.0012
mean			0.0019	0.0030

Conclusions:

LC0 (96h) = 0.0030 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 zebra fish (Brachydanio rerio) was determined in a 96-hour semi-static test with a daily test medium renewal according to the EU method C.1 (1992), and the OECD guideline 203 (1992).

A limit test was performed in accordance with the guidelines to demonstrate that the test item has no toxic effect on the test organisms up to and including the highest test item concentration which could be dissolved or very finely dispersed in test water. Due to the very low water solubility of the test item, supersaturated stock dispersions of the test item with a loading rate of nominal 100 mg/l were continuously stirred at room temperature in the dark over 7 days. Then, the dispersions were filtered. These filtrates were freshly prepared at each test medium renewal during this semi-static test. The undiluted filtrate with the maximum concentration of dissolved and very finely dispersed test item was used as the only test medium.

Additionally, a control was tested in parallel.

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 0.0039 and 0.0029 mg/l at the start of the test. After the test medium renewal period of one day, the measured concentrations slightly decreased to 0.0035 and 0.0018 mg/l. This slight decrease of the measured test item concentrations might have been caused by a precipitation of the finely dispersed test item, or due to a loss by adsorption onto the glass surfaces. All biological results are related to the mean measured test item concentration of 0.0030 mg/l (calculated as the average over all measurements in the undiluted filtrate during the test period).

In the control and in the undiluted filtrate, no mortality or other signs of intoxication were determined at the test fish during the test period of 96 hours.

Therefore, the 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours), and the 96-hour LC0 to zebra fish were determined to be at least 0.0030 mg/l (loading rate of 100 mg/l). The 96-hour NOEC and the 96-hour LC0 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 96-hour LOEC (lowest concentration with toxic effects), the 96-hour LC50 and the 96-hour LC100 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.

Description of key information

Highest concentration, which could be dissolved or very finely dispersed: 0.003 mg/l

EC0 (96h) = 0.003 mg/l

EC50 (96h) > 0.003 mg/l

Additional information

The toxicity to fish upon acute exposure was assessed using available data on test substance and on read across substances, i.e. Similar Substance 01 and 02. These are stuctural isomers of target substance, differing only for the position of -CN groups. A detailed read across justification is attached in section 13.

A 96 -hour semistatic study conducted on Similar Substance 02 was taken as key study, based on a sample purity of ca. 92 % and a detailed description of experimental procedures and results. It was conducted as limit test at a concentration of 0.003 mg/l, corresponding to the highest concentration that could be dissolved or very finely dispersed. No effects in terms of mortality or toxic symptoms were noted.

In a 96 -hour test on Similar Substance 01 (purity ca. 100 %), concentrations well-above the solubility were tested, namely 100, 300 and 1000 mg/l. No description of the solution was available in the study report; however, undissolved material was likely present. No effects in terms of mortality were reported.

In a 96 -hour static test on OB 199, concentrations between 1 and 300 mg/l were tested. Sample purity was ca. 42.4 % and no information on the other components was available. No mortality was seen up to 100 mg/l, while all fish died at the next dose of 300 mg/l. Accordingly, a LC0 of 100 mg/l and LC100 of 300 mg/l were established; a LC50 of 173 mg/l was derived.

Due to the low purity of the sample, compared with the other studies, and the lack of information on the composition of test material, this result was only taken as a qualitative indication of low toxicity towards fish.