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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 - 30 September 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study generally well performed and written. Method of analysis based on calibration curve from only two data points which is doubtful, no LOQ given, no data on validation of method. Study is a combined range-finder & limit test.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

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
AKZO Nobel trade name: PDTN
Chemical name : Acetonitrile, 2,2',2",2'"-(1,3-propanediyldinitri1o)tetrakis-(9 CI)
CAS-NO : 110057-45-9
Batch: JNN98038
Purity: 99.2% (+/- 1.0), CoA attached to report
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 1 June 2000
Stability in water: Not indicated

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
water solubility 1.66 g/L (flask method)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the final limit test samples were taken from 100 mg/l and the control for analysis.
Sampling: Frequency at t= 0 h and t= 48 h. Volume 10 ml from the approximate centre of the test vessels.
Storage The samples not analysed on the day of sampling were stored in a deep-freeze.
Additionally, reserve samples of 10 ml were taken. These samples were stored in a deep-freeze for a maximum of three months after delivery of the
draft report, pending on the decision of the sponsor for additional analysis, or until finalisation of the report.

All samples not analyzed on the sampling day were stored in a deep freeze. On the day of analysis, the frozen samples were defrosted
at room temperature. The entire volume of each sample (10 ml) was transferred quantitatively into a 25 ml test tube
using blank test medium (ISO-medium). 10 ml dichloromethane was added to the test tube.
Both phases were mixed vigorously for half a minute whereafter the organic layer was taken.
Again 10 ml dichloromethane was added to the test tube and extraction was repeated. For each
sample, the first and second organic layers were combined for analysis. The resulting solutions
were diluted using dichloromethane to obtain concentrations within the calibration range.

During the combined range finding/limit test, the recovery of the extraction procedure was verified using blank test medium (ISO-medium)
fortified with PDTN. Therefore, a solution of PDTN was prepared in acetone. An aliquot of this
solution (< 100 ul) was added to 10 ml IS0 medium (in triplicate). These control samples were
extracted with dichloromethane in the same way as the test samples.

Test solutions

Vehicle:

no

Details on test solutions:

The water solubility of PDTN was determined to be 1.66 g / l using the flask method (NOTOX Project 234877). A pretest was performed to examine the solubility of the test substance in the test medium. The pretest showed that PDTN was not easily soluble in the test medium or in any organic solvents. In the final limit test a solution was prepared at 100 mg/l. Magnetic stirring for 69 hours was used to ensure complete dissolving of the test substance.

ISO-medium: the following chemicals (analytical grade) are dissolved in freshly prepared ultra-pure water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ionexchange cartridges Milli-Q water; Millipore Corp., Bedford, Mass., USA) (mg/l):
CaCl2.2H20 293.8, MgSO4.7H20 123.3, NaHCO3 64.8, KCl 5.8

80 mL of test medium was put in 100 mL all glass vessels.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

1st instar daphnia magna: For the test selection of young daphnia with an age of < 24 hours.
Daphnia magna (Crustacea, Cladocera) (Straus,1820)
Validity: Frequent inspection of the cultures with respect to number of young, appearance of young and parental daphnia and global feeding behaviour.
Batch start: With newborn animals, i.e. less than 3 days o l d , by placing about 250 of them into 10 litres of medium in an all-glass culture vessel .
Maximum age of cultures: 4 weeks. Renewal: After 7 days of cultivation half of the medium twice a week.
Feeding: Daily, a suspension of fresh water algae

Test medium : M7, as prescribed by Dr. Elendt-Schneider (Elendt, B . - P . , 1990: Selenium deficiency in Crustacea. An ultrastructural approach to
antenna1 damage in Daphnia magna Straw. Protoplasma 154, 25-33):
- ISO-medium: the following chemicals (analytical grade) are dissolved in freshly prepared ultra-pure water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ionexchange cartridges Milli-Q water; Millipore Corp., Bedford, Mass., USA) (mg/l):
CaCl2.2H20 293.8, MgSO4.7H20 123.3, NaHCO3 64.8, KCl 5.8
- Medium M7; trace elements, macro nutrients and vitamines added to freshly prepared ISO medium.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

None

Test conditions

Hardness:

250 mg/L as CaCO3

Test temperature:

21.0-21.3 C

pH:

8.0 - 8.3

Dissolved oxygen:

8.7 - 8.9 mgO2/L

Nominal and measured concentrations:

0.1-1.0-10-100 mg/L

Details on test conditions:

10 daphnids in 0.1-1.0 and 10 mg/L dose groups (potential range finding)
20 daphnids in control and 100 mg/L dose groups

10 daphnids per vessel containing 80 mL medium, 16 h photoperiod, no feeding and aeration during exposure.
Introduction of daphnids within 0.5 hours after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

No mortality in control and exposed groups.
No sublethal effects observed in control or exposed groups.
Measured concentration in line with nominal concentration (< 10%).

Results with reference substance (positive control):

Reference substance performed as usual in the laboratory with an EC50 value of 0.52 mg/L after 48h (95%CL: 0.46-0.65) and 0.90 mg/L (0.75-1.2 mg/L) after 24h.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Study complies with validity criteria. No mortality or sublethal effects in static test. EC50 > 100 mg/L. Analytical section not solid.

Executive summary:

The study procedures described in this report were based on the IS0 Standard 6341, 1996-04-01. They were in compliance with the EEC directive 92169, Part C.2. 'Acute toxicity for Daphnia' and the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test', Adopted April 4, 1984. The water solubility of PDTN was determined to be 1.66 g/l using the flask method. However, a pretest showed that PDTN did not easily dissolve in the test medium. A range-finding test was combined with a limit test exposing Daphnia for a

maximum of 48 hours to a range of nominal concentrations from 0.1 to 100 mg/l. A stock solution o f 100 mg/l was stirred for 69 hours before the start of the test. The highest test concentration (100 mg/l) and the blankcontrol were tested in duplicate with 10 daphnia per vessel. Samples for analysis were taken at the start and the end of the test. Analysis of the samples taken during the limit test showed that the measured concentration was in agreement with nominal and remained stable during the test period. PDTN did not induce acute immobilization of Daphnia magna at 100 mg/l after 48 hours of exposure (NOEC). Hence, the EC50 was > 100 mg/l, the maximum

concentration tested.