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

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:

30 May - 29 October 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study is well performed and reported. However, statistics EbC questionable due to high number of points outside 95%CL and therefore the concluded (absolute) EbC50 value could be somewhat different, although actual concentrations were measured. Calculations and statistics are reproducible.

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 201 (Alga, Growth Inhibition Test)

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
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:

yes

Details on sampling:

During the final test samples were taken from three concentrations, i.e 10, 32 and 180 mg/L and the negative control.
Sampling: Frequency at t= 0 h and t=72 h, Volume 10 ml, Storage: the samples not analysed on the day of sampling were stored in a deep-freeze.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest
substance concentration but without algae and samples for analysis were taken at the start and the end of the test period.
Additionally, spare 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 delivery of the final report. Specification of the
samples analysed and the method of analysis are described in the appended Analytical Report.

All samples 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 M2-medium.
10 ml dichloromethane was added to the test tube and 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. If necessary, the solutions were diluted using dichloromethane to obtain concentrations within the calibration range.

Test solutions

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system were prevented as much as possible (e.g. film of the test substance on the water surface). 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. A pretest was performed to examine the solubility of the test substance in the test medium. This pretest showed that PDTN was not easily soluble in the test medium or in any organic solvents. In the range-finding test, test solutions were prepared by subsequent dilution starting with a stock solution of 1000 mg/l in IS0-medium, which was stirred for 91.5 hours prior to the start of the test.

In the EC50 test, stock solutions were prepared at 100 and 180 mg/l. The procedure of preparation of test solutions was kept as identical as possible. The stocks were stirred for 89.5 hours to ensure complete dissolving of the test substance. The 180 mg/l solution was tested as such and the 100 mg/l solution was used to prepare the other test concentrations. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, a volume of < 1 ml of an algal suspension was added to each replicate providing a cell density of l0^4 cells/ml.

Controls: Test medium without test substance or other additives (blank).
Replicates: 3 replicates of each test concentration. 6 replicates of the blank-control. 1 replicate of the highest concentration without algae.

Test organisms

Test organisms (species):

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

Details on test organisms:

Selenastrum capricornutum, strain: CCAP 27814. This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species. The results of the most recent reference test with potassium dichromate (Merck, Art. 4864) are appended to the report.

Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 +/- 2°C.
Preculture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2x10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21.2 - 23 C

pH:

8.1 - 8.4

Dissolved oxygen:

No data

Salinity:

Medium was M2 for which composition is given in report

Nominal and measured concentrations:

Nominal: 10-18-32-56-100-180 mg/L
Actuals: t=0h t=72h
nominal 10 10.1 10.5
nominal 32 34.5 34.0
nominal 180+algae 197 189
nominal 180-algae 189 191

Details on test conditions:

Test type: Static
Test vessels: 100 ml, all-glass
Milli-Q water: Tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges [Millipore Corp., Bedford, Mass., USA).
Cell density: An initial cell density of 1 x l0^4 cells/ml.
Test duration: 72 hours
Illumination: Continuously using TLD-lamps of 18 Watt (Philips, Spain) yielding 7500 - 8000 lux.
Medium: M2 according to ISO standard, Nov 1989
During incubation the algal cells were kept in suspension by continuous shaking
Vessels per concentration: 3
Vessels controls: 6

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95%CL: 30-120 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

129 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 54-311 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: EbC10

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95%CL: 11-46 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 13-74 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analytical results showed the measured concentrations were in close agreement with the nominal concentrations (<= 10%).
A dose response curve was obtained for both growth and growth rate reduction.

Based on the statistics used, an EbC50 was concluded at 60 mg/L. However, based on the mean of the triplicate samples, 60% inhibition was already obtained at 56 mg/L. All three measurements at this concentration level were outside the calculated 95% CL.
Based on the statistics used, an ErC50 was concluded at 129 mg/L.

Cell density increased by an average factor of > 16 within three days.

Results with reference substance (positive control):

The study with the reference substance was carried out in July 1998. The data are given in the report: EbC50= 0.92 mg/L and ErC50=1.4 mg/L.
Results were in agreement with the local historical control data (0.32 -1.8 mg/L and 0.56 -3.2 mg/L respectively).

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline (201, adopted 7 June 1984) were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth or inhibition of growth rate (ANOVA, Tukey test, Williams' test, TOXSTAT Release 3.0, September 1989, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in 'A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines by S. Pack, August 1993. Calculation of the EC50 and EC10 values was based on linear regression analysis of the percentages of growth inhibition or the percentages of growth rate reduction versus the logarithms of the corresponding test concentrations.

Any other information on results incl. tables

Inhibition algal growth and growth rate

Conc substance (in mg/L)	% inhibition growth	% inhibition growth rate
10	0.5	-0.4
18	-2.7	-1.1
32	19.4	5.0
56	59.8	24.4
100	76.1	46.0
180	85.3	59.5

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Basically, the study was performed and reported well, according to guideline and GLP. The study meets the validity criteria according to the guideline.Based on the statistical analysis, the EbC50 value is 60 mg/L, the ErC50 value is 129 mg/L and the lowest NOEC (growth inhibition) is 18 mg/L. However, looking at the figure for cell growth inhibition, the conclusion on EbC50 and ErC50 is not that evident.
The measured growth inhibition values of 10 and 56 mg/L are all outside the 95% Confidence Interval, raising some doubt about the validity of the interval or statistics used. The data of 10 mg/L have not been considered when calculating the interval for both growth and growth rate, but inclusion of this group will not give a different result since effects were absent and not different from the 18 mg/L group. EbC50 value should be treated with caution.

Executive summary:

The study procedures described were based on the EEC Directive 92169, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7, 1984; and IS0 Standard 8692, First edition, 15 November 1989. The water solubility of PDTN was determined to be 1.66 g /L using the flask method (NOTOX Project 234877). However, a pretest showed that PDTN did not easily dissolve in the test medium. After a range-finding test, a final test was performed exposing exponentially growing algal cultures to PDTN concentrations ranging from 10 to 180 mg/l, increasing with a factor of 1.8. The initial cell density was l0^4 cells/ml. The total test period was 72 hours. Samples for analysis were taken at 10, 32 and 180 mg/l at the start and the end of the test. Analysis of the samples showed that the measured concentrations were in agreement with nominal and remained stable during the test period. PDTN affected cell growth of the fresh water algae species Selenastrum capricornutum significantly at 32 mg/l and higher. The EC50 for cell growth inhibition (EbC50 :0-72h) was 60 mg/l with a 95% confidence interval ranging from 30 to 120 mg/l. The EC10 for cell growth inhibition (EbC10 :0-72h) was 22 mg/l with a 95% confidence interval ranging from 11 to 46 mg/l. The EC50 for growth rate reduction (ErC50 :0-72h) was 129 mg/l with a 95% confidence interval ranging from 54 to 311 mg/l. The EC10 for growth rate reduction (ErC10 :0-72h) was 31 mg/l with a 95% confidence interval ranging from 13 to 74 mg/l. The NOEC was 18 mg/l for cell growth inhibition (NOEbC) and 32 mg/l for growth rate reduction (NOErC) based on the Williams test (P=0.05).