Adiponitrile

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 08APR1994 to 09JUN1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to GLP and an internationally recognized guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Adiponitrile

EC Number:

203-896-3

EC Name:

Adiponitrile

Cas Number:

111-69-3

Molecular formula:

C6H8N2

IUPAC Name:

hexanedinitrile

Details on test material:

- Name of test material (as cited in study report): Adiponitrile
- Physical state: liquid
- Storage condition of test material: ambient temperature and protected from light.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Adiponitrile was measured in both the control treatment and the treatment exposed to 100 mg/L. Four samples were taken at test initiation and completion in each treatment, but only two were analysed (the two others were stored in a refrigerator in case further analyses were required).
- Sampling method: No data
- Sample storage conditions before analysis: The samples were analysed immediately after collection.

Test solutions

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: On the day of the test, an initial stock solution of 10000 mg/L was prepared as follow: 10.000 g of the test substance was weighted into a 50-mL bécker borosilicate glass flask. The content of this recipient was then transferred to a 1 liter-volumetric borosilicate glass flask and completed with test medium. The test solutions were prepared by diluting the stock solution with the test medium in order to get the desired experimental conditions.
- Controls: yes (test medium without test item)
No further data

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: CCAP number 278/4.
- Source (laboratory, culture collection): Axenic, uni-algal slope cultures from Culture Centre for Algae and Protozae (CCAP), Freshwater Biological Association, Ferry House, Far Sawrey, Ambleside, Cumbria.
- Age of inoculum (at test initiation): No data
- Method of cultivation: Initiatlly liquid cultures for the test inoculum were established by inoculation of OECD mineral salts medium with cells aseptically removed from slope cultures. Subsequently, subcultures were prepared from these liquid cultures by the aseptic transfer of aliquot into fresh OECD medium, which was then incubated for several days, until the required cell density had been achieved.

ACCLIMATION
No data

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

No data

Test temperature:

The temperature of the incubator ranged from 20.8 to 25.8°C during the test. The temperatures of the contents of control and test flasks at the start of the test were 22.0 and 21.9°C respectively; after 72 hours, temperatures ranged from 23.6 to 23.9°C.

pH:

The pH of the contents of the control and test flasks measured at the start of the test were both 8.0; after 72 hours pH ranged between 7.9 and 9.4.

Dissolved oxygen:

No data

Nominal and measured concentrations:

The nominal concentration was 100 mg/l (mean measured concetration of 97.4 mg/l).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed: no data
- Material, size, headspace, fill volume: 250 mL conical flasks containing 100-mL (50 mL OECD medium + 40 mL inoculum + 10 mL of OCED medium containing the test substance)
- Aeration: No data
- Initial cells density: 1 x 10E4 cells/mL
- Control end cells density: 165,85 x 10E4 cells/mL (mean of the 6 control replicates)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: OECD mineral salts medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD mineral salts medium
- Resistivity: 18 Megohm/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: the temperature and pH were measured at the initiation and completion of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: yes, the test vessels were sterilised by autoclaving (120°C for 15 minutes)
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: approximately 10125 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Algal cell density as detrmined each day of the test. 3-mL samples were removed from each flask and stored in numbered bottles at 4°c until required. the number of cells in each sample was determined using a haemocytometer; estimates of cell numbers were based on the means of 4 or 8 individual cells counts. Growth rate was then calculated using the following equation:
µ = (ln Nn - ln N1)/(tn - t1)
where: Nn = cells number at time tn, N1 = cells number at time t1, t1 = beginning of the test, tn = n hours or days after the beginning of the test.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 97.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 97.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Applicant's summary and conclusion

Conclusions:

Exposure to Adiponitrile at a test concentration of 97.4 mg/l did not cause a significant reduction (p>0.05) in either the average specific growth rate or biomass of Selenastrum capricornutum cultures. Thus, the no-observed-effect concentrations (NOECs) for growth rate and biomass were not identified but must be >97.4 mg/l. The 50% effect concentrations for average specific growth rate (ErC50) and mean biomass (EbC50) could not be computed.

Executive summary:

Because the classification criteria for aquatic toxicity is based on an EC50 value (<100 mg/l would be considered hazardous), this NOEC can only be used to validate the classification of non-hazardous because it can reasonably be assumed that an EC50 cannot be reached at <100 mg/l.