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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September - 2 October 1998
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
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. Deviation from guideline wrt oxygen levels did not influence study outcome. Limited data on fish supplier and no data on health status of batch of fish.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Oxygen levels below range specified in guideline. Aeration required and installed on Day 1.
GLP compliance:

Test material

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:
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, t= 24 h and t= 96 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 were stored in a deep freeze. Prior to extraction, 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.

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 sample.

Test solutions

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, aerated until the dissolved oxygen concentration had reached saturation and the pH had stabilized. The medium was formulated using Milli - Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
Ca2+ 80 mg/l
Mg 2+ 12mg/l
Na+ 15 mg/l
K+ 3 mg/l
Cl- 145 mg/l
SO42- 49 mg/ I
HC03- 47 mg/l

4 L of test medium was put in 5 L aquaria.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Zebra-fish (Danio rerio, Teleostei, Cyprinidae)
Origin: Charles River Aquatics, Someren , The Netherlands.
Mean length Limit test: 2.58 +/- 0.09 cm
Mean weight Limit test: 0.32 +/- 0.04 g
Feeding : Daily w ith Trouvit. No feeding from 48 hours prior to the test and during the total test period.

7 fish per concentration and controls.
Loading: 0.56 g fish/litre, i.e. 7 fish per 4 litres of test medium.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
Post exposure observation period:

Test conditions

Test medium: 250 mg/L CaCO3
Test temperature:
20.7 - 21.3 C
Dissolved oxygen:
At start of test9.0 mg/L dropping to 4.7 at day 1 after which continuous aeration was installed so oxygen levels increased again to 8.4 - 9.0 mgO2/L .
Nominal and measured concentrations:
Nominal: 100 mg/L
Actual: 107-109 mg/L
Details on test conditions:
5 L glass aquaria were used in which 7 fish were introduced after preparation of the test solutions (4L).
16h light period.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
96 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
meas. (not specified)
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.
Oxygen levels below range specified in guideline at day 1 for control and exposed groups, instalment of aeration solved the problem.
Results with reference substance (positive control):
Reference substance performed as usual in the laboratory with a LC50 value of 0.30 mg/L after 48h (95%CL: 0.28-0.39)

Applicant's summary and conclusion

Validity criteria fulfilled:
Oxygen levels proved non-lethal
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 EEC directive 92/69, Part C.1. "Acute toxicity for fish'; and the OECD guideline No. 203: Fish Acute Toxicity Test', Adopted 17 July, 1992. The water solubility of PDTN was determined to be 1.66 g/l using the flask method. However, a pre-test showed that PDTN did not easily dissolve in the test medium. A limit test was performed exposing seven fish to a solution of 100 mg/l. The test solution was stirred for 69 hours before the fish were introduced. Samples for analysis were taken at the start, after 24 hours and at the end of the test. Analysis of the samples taken during the final limit test showed that the measured concentration was in agreement with nominal and remained stable during the test period. No visible effects on zebra-fish were recorded. Hence, the LC50 of PDTN for fish was > 100 mg/l, the maximum concentration tested.