Benzene-1,3-dicarbonitrile

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:

28 February 2001 - 26 March 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study conducted by Japanese laboratories at the request of Japanese government. Report is translated by approved translation company.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

1) Name Isophthalonitrile
2) Chemical Name 1,3-Dicyanobenzene
3) CAS No. 626-17-5
5) Molecular Formula C8H4N2
6) Molecular Weight 128.13
7) Boiling Point 161.5°C *1
8) Solubility Soluble in nearly all organic solvents except for petroleum ethers, soluble in water when heated*2
9) Stability Readily sublimated*2
10) Lot No. Not available
11) Purity ≥ 98.0% (GC)*4
12) Vendor Not available
13) Supplier Products Safety Technology Center, Ministry of Economy, Trade and Industry
14) Confirmation of Test Substance
The infrared absorption spectrum measured at the Kurume office was analyzed, and it was confirmed that there were no inconsistencies in the structure of the test substance.

15) Storage Conditions and Confirmation of Stability Under Storage Conditions

(1) Storage Conditions
During testing, the test substance was stored in a cool, dark place.

(2) Confirmation of Stability
The infrared absorption spectrum of the test substance was measured before the beginning of exposure and after the end of exposure, and the stability was confirmed.

Analytical monitoring:

yes

Details on sampling:

The test substance concentration was measured at the start of exposure and before the water change. The test solution for measurement was sampled at the start of exposure from the preparation container, and from the middle layer of one of the test containers before water replacement. In the case of the saturated solutions, the test substance concentration was confirmed during preparation.

Vehicle:

no

Details on test solutions:

Test Solution Preparation Method

The test substance equivalent to 500 mg/L was added to test water heated in a water bath at about 80°C, and stirred with a stirrer while irradiating the solution with ultrasonic waves for five minutes. After cooling to about 20°C., the precipitate was filtered through a 0.45 μm membrane filter (from Millipore) to obtain a saturated solution. The concentration of the saturated solution was confirmed before preparing the test solution.

Saturated Solution Preparation

The saturated solution was used directly as the test solution or diluted by mixing in the required amount of test water by stirring. This was prepared for each test substance concentration group, and divided among the test containers.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna (Daphnia magna Clone A). Larva was subcultured at the Kurume office from descendants of Daphnia magna (Clone A) procured from the University of Sheffield in the United Kingdom (address: Sheffield S10 2UQ, UK).
The larvae produced by a 28-day-old adult group were used within 24 hours of hatching. Random extraction was performed.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Swimming inhibition and symptoms were observed and recorded at the beginning of exposure and after 24 and 48 hours of exposure.

Post exposure observation period:

If the organisms could not swim for 15 seconds after gently disturbing the test solution, swimming was considered inhibited.

Hardness:

Not measured.

Test temperature:

20.2 to 20.4°C

pH:

7.6 to 7.7

Dissolved oxygen:

7.6 to 8.7 mg/L

Salinity:

Not measured.

Conductivity:

Not measured.

Nominal and measured concentrations:

The test substance concentrations in the test solutions using the saturated solution and the 1/4 and 1/2 diluted solutions were 430, 217, and 108 mg/L, respectively, at the beginning of exposure, and 424, 221, and 109 mg/L, respectively, before the water was changed.
The test concentrations and common ratios were determined from preliminary test results

Details on test conditions:

Testing Conditions
Method
A chemical bath method was used in which the test organism was exposed to a test solution containing the test substance. The test was conducted using the semi-static method in which all of the test solution was changed after 24 hours.

Period
48 Hours

Concentrations
The test was performed on three concentration groups [saturated solution, 1/2 and 1/4 diluted solutions of the saturated solution (common ratio 2.0)] and a control group. The test concentrations and common ratios were determined from preliminary test results. The concentrations in the tables are the averages of the measured values.

Control Group
Only test water was used.

Number of Test Organisms
20 organisms were used per test group (5 organisms per test container x 4 test containers).

Test Solution
The test was conducted using 800 mL per test group (200 mL per test container × 4 test containers).

Test Solution Preparation Method

The test substance equivalent to 500 mg/L was added to test water heated in a water bath at about 80°C, and stirred with a stirrer while irradiating the solution with ultrasonic waves for five minutes. After cooling to about 20°C., the precipitate was filtered through a 0.45 μm membrane filter (from Millipore) to obtain a saturated solution. The concentration of the saturated solution was confirmed before preparing the test solution.

Saturated Solution Preparation

The saturated solution was used directly as the test solution or diluted by mixing in the required amount of test water by stirring. This was prepared for each test substance concentration group, and divided among the test containers.

Observations and Measurements

State of Test Organisms
Swimming inhibition and symptoms were observed and recorded at the beginning of exposure and after 24 and 48 hours of exposure. If the organisms could not swim for 15 seconds after gently disturbing the test solution, swimming was considered inhibited.

State of Test Solution
The state of the test solution was observed and recorded at the start of exposure (during preparation) and before the water change.

Water Quality
The dissolved oxygen concentration, the pH, and the water temperature were measured in a test container (test solution) for water quality measurements prepared separately but using the same preparation method as the test solution used in the test, and the same measurements were made using one of the four test containers in each test group before changing the water. The dissolved oxygen concentration was measured using dissolved oxygen meter No. 58 (from Yellow Springs Instrument Co., Ltd.), the pH was measured using a glass electrode type hydrogen ion concentration meter HM-14P (from Toa Denki Kogyo), and the water temperature was measured using a verified glass rod thermometer.

Concentration of Test substance in the Test Solution

Water Sampling and Analysis
The test substance concentration was measured at the start of exposure and before the water change. The test solution for measurement was sampled at the start of exposure from the preparation container, and from the middle layer of one of the test containers before water replacement. In the case of the saturated solutions, the test substance concentration was confirmed during preparation.

Preparation of Reference Solution
A reference solution for determining the concentration of the test substance in the analyzed samples was prepared in the following way. 100 mg of the test substance was accurately weighed using an electronic analysis balance and dissolved in acetonitrile to prepare a 1,000 mg/L test substance solution. This solution was diluted with acetonitrile to prepare a 100 mg/L test substance solution. This was diluted to acetonitrile/medium 1/1 (v/v) to obtain a 5.00 mg/L reference solution.

Environmental Conditions:
Observations and Measurements - Performed at 20° ± 1°C.
Illumination - Artificial lighting was provided by indoor lighting for 16 hours a day.
Feeding - There was no feeding during the exposure period.
The 48-hour EC50 in the acute swimming inhibition test conducted regularly using a reference substance (potassium dichromate, reagent special grade, Wako Pure Chemical Industries, Ltd.) was 0.248 mg / L. This value was within the 48-hour EC50 allowable range for the reference substance at the Kurume office (mean ± 2 x standard deviation 0.0909-0.316 mg/L) [average standard deviation = 0.203 ± 0.056 mg/L].

Evaluation of NOEC (No Observed Effect Concentration)
NOEC (No Observed Effect Concentration) is defined as the highest concentration group in which no symptoms were observed compared to the control group during the exposure period.

Handling Numerical Values
The numerical values were rounded off in accordance with JIS Z8401-1999 Rule B.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 427 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

· Test substance concentration during testing: The measured concentration before the water change was kept within ±20% of the measured concentration at the time of preparation.
· 48-Hour EC50: > 427 mg/L
· NOEC: 108 mg/L
· OECD hazard classification: None (Not Applicable)

EC50
The 24- and 48-hour EC50 were > 427 mg/L for isophthalonitrile on Daphnia magna.

Swimming Inhibition Rate
The lowest concentration for 100% swimming inhibition at 48 hours was not obtained in the concentration range of this study. The maximum concentration with 0% swimming inhibition was 219 mg/L. The swimming inhibition rate in the control group during the exposure period was 0% and met the efficacy standard (not exceeding 10%).

Observation Results Such as Symptoms
The following observations are all based on comparison with the control group. The NOEC during the exposure period was 108 mg/L. Symptoms observed during the exposure period were reduced activity and swimming inhibition.

Test Solution Observations and Measurement Results

State of Test Solution
The test solution was colorless and transparent when prepared. Before the water change, the saturated solution had suspended matter on the water surface and precipitate settled on the bottom of the container. Other test groups were as colorless and transparent as during preparation.

Water Quality of the Test Solution
The dissolved oxygen concentration in the test substance concentration groups measured at the start of exposure and before the water change was 7.6 to 8.7 mg/L, the pH was 7.6 to 7.7, and the water temperature was 20.2 to 20.4°C. The dissolved oxygen concentration satisfied the efficacy standard (at least 60% of the saturated concentration at the test water temperature*).

*(Saturated dissolved oxygen concentration at 19-21°C: 9.01-8.68 mg/L, JIS K 0102-2000)

Test Substance Concentrations in the Test Solutions
The test substance concentrations in the test solutions using the saturated solution and the 1/4 and 1/2 diluted solutions were 430, 217, and 108 mg/L, respectively, at the beginning of exposure, and 424, 221, and 109 mg/L, respectively, before the water was changed. Table 5 shows the measured test substance concentrations.

Test Solution Observations and Measurement Results
In the 48-hour acute swimming inhibition test on isophthalonitrile using Daphnia magna, the test substance concentration during preparation of the test solution was maintained until the water was changed. The 48-hour EC50 for Daphnia was > 427 mg/L. The numerical value is > 100 mg/L, and because it falls outside of the OECD hazard classification, isophthalonitrile has been evaluated as having low toxicity to Daphnia magna.

The following conclusions have been reached based on the test results.
· Test substance concentration during testing: The measured concentration before the water change was kept within ±20% of the measured concentration at the time of preparation.
· 48-Hour EC50: > 427 mg/L
· NOEC: 108 mg/L
· OECD hazard classification: None (Not Applicable)

Environmental factors that may have influenced the reliability of test results
There are no such factors.

Results with reference substance (positive control):

Within parameters.

Validity criteria fulfilled:

yes

Conclusions:

In the 48-hour acute swimming inhibition test on isophthalonitrile using Daphnia magna, the test substance concentration during preparation of the test solution was maintained until the water was changed. The 48-hour EC50 for Daphnia was > 427 mg/L. The numerical value is > 100 mg/L, and because it falls outside of the OECD hazard classification, isophthalonitrile has been evaluated as having low toxicity to Daphnia magna.

Executive summary:

A 48-hour acute swimming inhibition test on isophthalonitrile was conducted using Daphnia magna.

The test was performed using 20 organisms per test group in three concentration groups [saturated solution, 1/2 and 1/4 diluted solutions of the saturated solution (common ratio 2.0)] and a control group using a semi-static method in which all of the test solution was replaced after 24 hours at a water temperature of 20 ± 1°C.

As a result, the test substance concentrations in the saturated solution and in the 1/2 and 1/4 diluted solutions of the saturated solution were 430, 217, and 108 mg/L, respectively, at the beginning of exposure, and 424, 221, and 109 mg/L, respectively, before the water was changed. Note thatthe average of the measured values was used to calculate the results.

The 24-hour and 48-hour EC50 of isophthalonitrile was > 427 mg/L.The minimum concentration for 100% swimming inhibition at 48 hours was not obtained from the concentration range in this study. The maximum concentration with 0% swimming inhibition concentration was 219 mg/L and the NOEC (no observed effect concentration) was 108 mg/L.

Description of key information

A 48-hour acute swimming inhibition test on isophthalonitrile was conducted using Daphnia magna.

The test was performed using 20 organisms per test group in three concentration groups [saturated solution, 1/2 and 1/4 diluted solutions of the saturated solution (common ratio 2.0)] and a control group using a semi-static method in which all of the test solution was replaced after 24 hours at a water temperature of 20 ± 1°C.

As a result, the test substance concentrations in the saturated solution and in the 1/2 and 1/4 diluted solutions of the saturated solution were 430, 217, and 108 mg/L, respectively, at the beginning of exposure, and 424, 221, and 109 mg/L, respectively, before the water was changed. Note thatthe average of the measured values was used to calculate the results.

The 24-hour and 48-hour EC50 of isophthalonitrile was > 427 mg/L.The minimum concentration for 100% swimming inhibition at 48 hours was not obtained from the concentration range in this study. The maximum concentration with 0% swimming inhibition concentration was 219 mg/L and the NOEC (no observed effect concentration) was 108 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

427 mg/L