Acetonitrile, 2,2',2'',2'''-(1,3-propanediyldinitrilo)tetrakis-

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 June 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study well performed and reported according to GLP and guideline. No validation of test concentration by actual measurement (stock or test solutions), but very likely nominal = actual since other ecotox tests were performed with higher (actual) concentrations at the same lab in the same year.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Principles of method if other than guideline:

No measured concentrations

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-330-7

EC Name:

-

Cas Number:

110057-45-9

Molecular formula:

C11H14N6

IUPAC Name:

2-({3-[bis(cyanomethyl)amino]propyl}(cyanomethyl)amino)acetonitrile

Details on test material:

Identification: PDTN
Description: White crystalline solid
AKZONobel trade name: PDTN
Chemical name: Acetonitrlle, 2,2',2",2"'-(1,3-propanediyldinitri1o)tetrakis-(9 CI)
CAS - No.: 110057-45-9
Batch: JNN98038
Purity: 99.2 +/- 1% m/m (see Certificate of Analysis attached to report)
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 June 2000
Stability in water: Not indicated

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

Test solution was prepared in duplicate, wherby 50.9 and 49.8 mg were dissolved in and up to 300 ml milliQ water, after which 200 ml activated sludge was added, together with 16 ml of synthetic sewage feed.
4 Controls were prepared as the test substance (but without test substance).
A reference substance series with 3.2, 10 and 32 mL of reference stock solution (124.7 mg 3,5-dichlorophenol in 250 mL, with a pH of 7.8) was added to 16 mL of synthetic sewage feed, 200 mL of activated sludge and made up to a total volume of 500 mL with milli-Q water.

Consequently, test concentrations were ca. 100 mg/L and reference substance concentrations were 3.2, 10 and 32 mg/L.

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

Origin of the sludge was Municipal sewage treatment plant: 'Waterschap de Maaskant' at 's-Hertogenbosch, The Netherlands.
The sludge was coarsely sieved, washed and diluted with tap water. A small amount was weighed and dried at 105C.
From the result, the amount of suspended solids was calculated at 3.8 g/L.
The batch of sludge was used on subsequent days.
50 mL of synthetic sewage feed was added to each litre of activated sludge at the end of each working day.
The sludge was kept aerated at test temperature until use. Before use the pH was checked (7.7)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

30 min

Post exposure observation period:

None

Test conditions

Hardness:

No specific data

Test temperature:

Test medium: 19.7-20.4 C

pH:

7.1-7.3

Dissolved oxygen:

Oxygen reduction is parameter of investigation

Salinity:

Details on synthetic sewage feed are given in report.

Nominal and measured concentrations:

Nominal:
PDTN: 100 mg/L
3,5-dichlorophenol: 3.2-10-32 mg/L

Details on test conditions:

Test vessels: All glass, 500 ml beakers and 300 ml oxygen bottles.

A well mixed sample of the contents was poured into a 300 ml oxygen bottle, and the flask was sealed with an oxygen electrode connected to a recorder forcing the air not to remain in the vessel. Oxygen consumption was measured and recorded for approximately 10 min. During measurement the sample was not aerated but continuously stirred on a magnetic stirrer.
The pH was determined in the remaining part of the reaction mixture.
This procedure was repeated for the duplicate concentration. In each test series two controls without test substance were tested, one at the start and one at the end.

Reference substance (positive control):

yes

Remarks:

3,5 dichlorophenol

Results and discussion

Effect concentrations

Duration:

30 min

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The mean respiration rate of the controls did not differ more than 3%. No significant inhibition in respiration rate of the sludge was recorded at approx. 100 mg/L

Results with reference substance (positive control):

EC50 of 3,5 dichlorophenol was 9 mg/L, which is acceptable according to the guideline.

Any other information on results incl. tables

Concentration (mg/L)	Oxygen consumption (mg O2/l/hr)	% inhibition
Control 1	33	-
Control 2	34	-
Control 3	34	-
Control 4	34	-
PDTN     100	32	6
PDTN     100	39	-9
3,5 DCP     3.2	25	25
10	17	49
32	6	82

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Study complies with guideline, GLP and validity criteria. Conclusions valid as nominal concentration is likely to be actual since other ecotox studies with PDTN performed at same lab in same year have measured actuals which are > 100 mg/L. Therefore the EC50 or even EC10 of PDTN for microorganisms from sewage treatment plants is > 100 mg/L.

Executive summary:

The influence of PDTN on the respiration rate of activated sludge was investigated after a contact time of 30 minutes.

The study procedure was based on OECD Guideline No. 209, adopted April 4, 1984 and EEC Directive 67/548 amended November 18, 1987 (871302), Part C.11, Publication No. L133, adopted May 30, 1988. Since PDTN was hardly soluble in water, PDTN was added quantitatively to the test vessels at a concentration of approximately 100 mg/l. This concentration was tested in duplicate. No significant inhibition in respiration rate of the sludge was recorded at approximately 100 mg PDTN per litre. The duplicate measurement confirmed the result of the first measurement. Therefore no further testing was needed. The respiration rates of the controls were within 15% of each other. The EC50 of the reference substance, 3,5-dichlorophenol, was 9 mg/l. Therefore, the test was considered to be valid. In conclusion, under the conditions of this present test PDTN was not toxic to waste-water bacteria at a concentration of approximately 100 mg/l.