Benzene-1,3-dicarbonitrile

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 February 2001 - 27 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 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

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 solution was measured at the beginning and at the end of exposure. The pH at the start of the exposure was measured in a test container prepared for water quality measurements, and the pH at the end of the exposure was measure in one of the three test containers. The pH was measured using a HM-14P glass electrode-type hydrogen ion concentration meter (from Toa Denki Kogyo). The temperature and illuminance in the culture apparatus were measured once per day during the exposure period. The temperature was measured using a verified glass rod thermometer, and the illuminance was measured using an SLX-1330 illuminometer (from Sansho).

Counting was performed using a particle counter (Coulter Counter Model Z1 from Coulter Electronics, Inc.) every 24 hours after the start of exposure for up to 72 hours. At the end of exposure, the state of the cells in all test groups was observed using a biological microscope (BH-2 from Olympus Optical Co., Ltd.).

Vehicle:

no

Details on test solutions:

The test was conducted on five concentration groups [100, 62.5, 39.1, 24.4, and 15.3 mg/L (1.6 common ratio) and a control group. Test substance concentrations and common ratios were determined based on preliminary test results.
The test was performed three times for each test group.

Control Group
A medium prepared using the same treatment as the test stock solution and an untreated medium were prepared at the same mixing ratios as the concentration groups.

Test organisms (species):

other: Selenastrum capricornutum

Details on test organisms:

Selenastrum capricornutum (ATCC 22662), the reason for selecting this species is because this is the standard species recommended in the OECD test guidelines.

The Kurume office subcultured Selenastrum capricornutum procured from the American Type Culture Collection (12301 Parklawn Drive Rockville, Maryland 20852-1776 U.S.A.). The 72-hour EC50 of the reference material from the lot used in the test (potassium dichromate, reagent grade, Wako Pure Chemical Industries, Ltd.) based on the area under the growth curve was 0.427 mg/L, and this value was within the allowable range for the reference substance using the same index at the Kurume office (mean ± 2 x standard deviation: 0.299-0.437 mg/L) [average standard deviation = 0.368 ± 0.035 mg/L].

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

As per the guideline

Post exposure observation period:

As per the guideline

Hardness:

Not measured.

Test temperature:

22.7 - 22.8°C

pH:

7.7 - 7.8 at start of exposure
7.7 - 10.4 at end of exposure

Dissolved oxygen:

Not measured.

Salinity:

Not measured.

Conductivity:

Not measured.

Nominal and measured concentrations:

15.3, 24.4, 39.1, 62.5, 100 mg/L

Details on test conditions:

Exposure Conditions
Method
A gyratory shaking culture was performed using the chemical bath method in which the test organism is exposed to a test solution containing the test substance.

Period
72 Hours

Concentrations
The test was conducted on five concentration groups [100, 62.5, 39.1, 24.4, and 15.3 mg/L (1.6 common ratio) and a control group. Test substance concentrations and common ratios were determined based on preliminary test results.

Number of Iterations
The test was performed three times for each test group.

Control Group
A medium prepared using the same treatment as the test stock solution and an untreated medium were prepared at the same mixing ratios as the concentration groups.

Testing Operations
Sterile operations were performed.

Amount of Test Solution
Each test group used 300 mL (100 mL × 3 test containers).

Environmental Conditions
Water Temperature
The water temperature was 23 ± 2°C.

Illumination
Continuous illumination was performed at an illuminance of about 4,000 to 5,000 lux.

Medium
The medium (OECD recommended medium) indicated in the OECD chemical test guidelines was used for both the preculture and the test. The composition of the medium is shown in Appendix-1. The medium was sterilized.

Number of Cells at the Start of Exposure
104 cells/mL

Inoculation Source
An algae culture solution containing cells in logarithmic growth phase planted from a preservation culture to preculture medium and then cultured for 3 days under the test conditions was inoculated into the test culture solution.

Test Stock Solution Preparation Method
The test substance was mixed with the medium at about 80°C, and then stirred with a stirrer and dissolved while being irradiated with ultrasonic waves for about one hour to prepare a 200 mg/L test stock solution. This was sterilized by filtration through a 0.45 μm membrane filter.

Test Solution Preparation Method
The required amount of test stock solution and medium were mixed together to prepare a test solution for each concentration group. This was prepared for each concentration group and divided among the test containers. In order to take into account the effects of ultrasonic irradiation, an appropriate amount of medium prepared in the same manner as the test stock solution was added so that the volume of the ultrasonically agitated liquid and the volume of the untreated liquid were the same in all test groups.

Observations and Measurements

Cell Concentration Measurement
Counting was performed using a particle counter (Coulter Counter Model Z1 from Coulter Electronics, Inc.) every 24 hours after the start of exposure for up to 72 hours. At the end of exposure, the state of the cells in all test groups was observed using a biological microscope (BH-2 from Olympus Optical Co., Ltd.).

Water Quality and Exposure Environment
The test solution was measured at the beginning and at the end of exposure. The pH at the start of the exposure was measured in a test container prepared for water quality measurements, and the pH at the end of the exposure was measure in one of the three test containers. The pH was measured using a HM-14P glass electrode-type hydrogen ion concentration meter (from Toa Denki Kogyo). The temperature and illuminance in the culture apparatus were measured once per day during the exposure period. The temperature was measured using a verified glass rod thermometer, and the illuminance was measured using an SLX-1330 illuminometer (from Sansho).

Concentration of Test substance in the Test Solution
Water Sampling and Analysis
Test substance concentrations were measured at the beginning and at the end of exposure. The test solution for the measurement at the start of exposure was collected from the container prepared for the water quality measurements, and the test solution for the measurement at the end of exposure was taken from one of the three test containers for each test group. These samples were mixed together and centrifuged to remove the algae.

Reference substance (positive control):

no

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

54.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

63.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

EC50
The isophthalonitrile in EbC50(0-72), ErC50(24-48) and ErC50(24-72) calculated based on the area under the growth curve, the 24-48 hour growth rate, and the 24-72 hour growth rate were 40.6 mg/L, 54.2 mg/L and 63.8 mg/L, respectively.

Growth Curves, Cell Observation Results, and NOEC of Each Test Group
The growth of the test algae in the control group showed logarithmic growth until the end of the exposure. At the end of exposure, it had grown to more than 117 times the initial number of cells. This met the efficacy criteria (proliferation of more than 16 times). In the 100 mg/L group, growth was suppressed significantly throughout the exposure period. Although logarithmic growth was observed in the 62.5 mg/L group, it showed a strong inhibition tendency overall. While inhibition was observed in the 39.1 mg/L group, logarithmic growth similar to that in the control group was shown. The 24.4 and 15.3 mg/L groups showed almost the same amount of growth as the control group.

In the cell observations, a larger number of swollen cells were observed in the 100 mg/L group than in the control group, and there was slightly more in the 62.5 mg/L group. In the other concentration groups, the cell state was almost the same as in the control group.

In the results of the significant difference test and the cell observation results, NOEC for the area under the growth curve, 24-48 hour growth rate, and 24-72 hour growth rate were 24.4 mg/L, 24.4 mg/L, and 39.1 mg/L, respectively.

Test Solution Observations and Measurement Results

State of Test Solution
The test solution was colorless and transparent at the time of preparation. At the end of exposure, the test solution was pale green in the 100 mg/L concentration group, light green in the 62.5 mg/L group, and green in other concentration groups due to the growth of algae.

Water Quality of the Test Solution and the Exposure Environment

The pH of the test solution in the test substance concentration groups was 7.7 to 7.8 at the start of exposure and 7.7 to 10.4 at the end of exposure. The temperature in the culture apparatus was 22.7 to 22.8°C, and the illuminance was 4,000 lux.

Test Substance Concentrations in the Test Solutions
The test substance concentrations in the test solutions was 96.8 to 99.5% of the set value at the start of exposure and 96.1 to 98.9% at the end of exposure.

Evaluation, Observations and Conclusions Based on the Test Results
In the algae growth inhibition test of selenastrum capricornutum by isophthalonitrile, the test substance concentration in the test solutions was maintained at the set value, and the EbC50(0-72), ErC50(24-48) and ErC50(24-72) for Selenastrum capricornutum were 40.6 mg/L, 54.2 mg/L, and 63.8 mg/L, respectively. These values are in the range of EC50:10-100 mg/L and the OECD hazard classification is Class: Acute III.

The following conclusions have been reached based on the test results.
· Test substance concentrations during testing:
The measured concentrations were 96.8 to 99.5% at the start of exposure and 96.1 to 98.9% at the end of exposure, and thus remained at the set concentration throughout the exposure period. Therefore, the test results are based on the set concentration.

Results with reference substance (positive control):

Not applicable.

Validity criteria fulfilled:

yes

Conclusions:

The measured concentrations were 96.8 to 99.5% at the start of exposure and 96.1 to 98.9% at the end of exposure, and thus remained at the set concentration throughout the exposure period. Therefore, the test results are based on the set concentration.

· EC50:
EbC50(0-72); 40.6 mg/L (37.5 ~ 43.9 mg/L)
ErC50(24-48); 54.2 mg/L (40.3 ~ 72.8 mg/L)
ErC50(24-72); 63.8 mg/L (56.3 ~ 72.2 mg/L)

· NOEC:
Area under the growth curve: 24.4 mg/L
24-48 hours growth rate: 24.4 mg/L
24-72 hour growth rate: 39.1 mg/L

Executive summary:

An isophthalonitrile algae growth inhibition test was conducted using Selenastrum capricornutum.

The test was conducted using a gyratory shaking culture (approx. 100 times per minute) on five concentration groups [100, 62.5, 39.1, 24.4 and 15.3 mg/L (1.6 common ratio)] and a control group. The exposure time was 72 hours, the culture temperature was 23 ± 2°C, and the artificial lighting was white fluorescent light (illuminance approx. 4,00 to 5,000 lux, continuous illumination).

As a result, the test substance concentration in the test solution was 96.8 to 99.5% of the set value at the start of exposure, and 96.1 to 98.9% at the end of the exposure. In other words, the set concentration was nearly maintained. Therefore, the test results were calculated based on the set concentration.

The EC50 (50% growth inhibitory concentration) of isophthalonitrile calculated based on the area under the growth curve, the growth rate over 24-48 hours, and the growth rate over 24-72 hours was 40.6, 54.2 and 63.8 mg/L, respectively. The no observed effect concentrations (NOEC) were 24.4, 24.4 and 39.1 mg/L, respectively.

Description of key information

An isophthalonitrilealgae growth inhibition test was conducted using Selenastrum capricornutum.

The test was conducted using a gyratory shaking culture (approx. 100 times per minute) on five concentration groups [100, 62.5, 39.1, 24.4 and 15.3 mg/L (1.6 common ratio)] and a control group. The exposure time was 72 hours, the culture temperature was 23 ± 2°C, and the artificial lighting was white fluorescent light (illuminance approx. 4,00 to 5,000 lux, continuous illumination).

As a result, the test substance concentration in the test solution was 96.8 to 99.5% of the set value at the start of exposure, and 96.1 to 98.9% at the end of the exposure. In other words, the set concentration was nearly maintained. Therefore, the test results were calculated based on the set concentration.

The EC50 (50% growth inhibitory concentration) of isophthalonitrile calculated based on the area under the growth curve, the growth rate over 24-48 hours, and the growth rate over 24-72 hours was 40.6, 54.2 and 63.8 mg/L, respectively. The no observed effect concentrations (NOEC) were 24.4, 24.4 and 39.1 mg/L, respectively.

 

Key value for chemical safety assessment

EC50 for freshwater algae:

40.6 mg/L

EC10 or NOEC for freshwater algae:

24.4 mg/L