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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28-10-2008 to 2-12-2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study is complete and GLP

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:

Test material

Details on test material:
- Name: PDTN
- chemical name: 1,3-propylenediaminetetraacetonitrile
- Purity / active ingredient: 98.5% (99.9 wt % dried)
- appearance: White solid/crystals
- water solubility: 1700 mg/L in water at 20 °C 7500 mg/L at 50 °C
- vapour pressure: 1.4 x 10-1 Pa
- Lot number: CFC-8976
- stability: In aqueous systems slight acidic pH is preferred for optimal stability (2.5- 5.5). It will however be considered stable in test scenario.
- storage until required: at room temperature away from light

Sampling and analysis

Analytical monitoring:
Details on sampling:
Samples of all the test concentrations were taken on preparation (fresh) and after changing (used) of the test solution.
The volume of the samples of the new test concentrations was 10 mL directly pipetted into a glass test bottle. For sampling after changing the test solutions, 1 mL was taken from every replicate and pooled, these pooled samples were filtered over a 0.45 µm GHP Acrodisc filter to remove the algae. The samples were directly analyzed or stored at room temperature in the dark.
Samples of new test solutions taken at the beginning of the test were analyzed and thereafter only after changing of the test solutions.

Test solutions

Details on test solutions:
The pH of the test solutions were all between 6 and 9 throughout the test and close to the value of M4 reconstituted water after each solution change. Only once the pH of the fresh test solution of the highest concentration was out of range. The control pH was 7.7, while the pH of the test concentration was only 7.1. Probably this was a technical error in the measurement because the pH measured in the stock solution used and in the control was in the proper range. This deviation is not considered to have had an impact on the integrity or quality of the study.
One control containing only test medium was included in the test.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: Dr .U. Noack-Laboratorien Kathe-Paulus- Str. 1, D-31157 Sarstedt, Germany) cultured in conformity with the relevant SOP. The animals used in the test were less than 24 hours old and were obtained from parent animals
reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use).

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
21 d

Test conditions

179-301 mg/L CaCO3
Test temperature:
20.5-21.6 C
Dissolved oxygen:
8.2-10.95 mg/L
Nominal and measured concentrations:
Nominal test concentrations: 0, 10, 32, 102.4, 327.7 and 1049 mg/L.
Mean measured test concentrations: 0, 12.5, 35.4, 107.6, 314.3 and 1092.7 mg/L
% of nominal: -, 125.4, 110.6, 105.0, 95.9, 104.2 %

Details on test conditions:
- Test vessel: 50 mL (nominal) glass beakers were used, containing approximately 50 mL of test solution and covered by glass plates during the test
- Type (delete if not applicable): open
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 3 times per week. This is not explicitely stated in the report but can be concluded from the chemical analysis as samples were taken at every renewal and all samples taken were analyzed.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

- Source/preparation of dilution water: Elendt M4 medium
- Culture medium different from test medium: no

- Adjustment of pH: no
- Photoperiod: 16 hours of ambient light per day, provided by fluorescent tubes
- Light intensity: 15 and 20 µmol/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : parent mortality, parent lenght and weight, number of young produced

- Results used to determine the conditions for the definitive study: The test concentrations were based on an available acute study where no effect up to 100 mg/L was observed. Therefore it was decided to perform an additional non-GLP acute screening test at 500 and 1000 mg/L. This test also showed no effect up to 1000 mg/L.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
21 d
Dose descriptor:
Effect conc.:
10 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
25.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
51 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
148.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
Chemical analysis:
At 327.7 mg/L after 13 and 15 days of testing the observed concentrations were 129.8 and 210.0 mg/L, respectively. This is a decrease of 60 and 36%, respectively, compared to the nominal concentration. At day 17 this test concentration was measured at 9% of the nominal concentration, but after reanalyzing it turned out to be normal (352.2 mg/L). Therefore additional chemical analysis was performed on three fresh samples. These additional measurements were found to be acceptably close to the nominal values and established that no errors in preparation / dilution were made. It is also worthy to note the anomalies mentioned were only observed on days that daphnia had died. The 327.7 mg/L replicate remained within acceptable limits on average and this slight fluctuation was not considered to have any impact on the outcome of the test. Furthermore the endpoints of interest were not at this concentration.

Parental mortailty:
One daphnid died in each of the following concentrations: 10 mg/L (day 8) and 102.4 mg/L (day 20). Two daphnids died in 32 mg/L (day 9 and 13). All daphnids died during the test in 327.7 and 1049 mg/L.

Other biological effects observed:
The first brood release was found to be weak in all test concentrations. At 10 and 32 mg/L the second brood was also found to be weak.
On one occasion in 102.4 mg/L (day 15) in total 5 dead young were found in two replicates. In the same test concentrations on day 20 some aborted eggs were seen in two replicates.
At the two highest test concentrations no young were produced throughout the test.
Reported statistics and error estimates:
The data on reproduction was tested for normality using Shapiro-Wilk’s test. The data were checked for homogeneity of variance using Bartlett’s tests. Analysis of variance was performed on the number of living neonates per parent using the Bonferroni t-test and verified with a second multiple comparison method, the Dunnett’s test.

Applicant's summary and conclusion

Validity criteria fulfilled:
Test is valid, the lowest Observed Effect Concentration (LOEC) was found to be 32 mg/L. Based on these statistical results the NOEC is 10 mg/L. An EC10 and EC50 for reproduction were determined as well they were 25.6 mg/L and 51.0 mg/L, respectively. The EC50 for parent survival was 148.6 mg/L.
Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211, 21st September 1998and EU guideline C.20 from Annex V of Directive 67/548/EEC.


The test criterion of toxicity used was reproductive capacity expressed as the number of neonates per daphnid per day.


The nominal concentrations used in the study were as follows: 0, 10, 32, 102.4, 327.7 and 1049 mg/L

All concentrations given refer to the technical product as supplied by the sponsor.


Analytical determinations of the test solutions were made on 13 occasions during the test. The concentrations were found to remain within 80 and 120% of the nominals for 32 to 1049 mg/L. Only 10 mg/L was measured higher on average than 120%. This was due to the intercept of the calibration curves used to calculate the concentrations measured. All calibration curves had an intercept between 0.88 to 4.19 mg/L. On lower test concentrations this intercept has an increasing effect as test concentrations are lower. As all other concentrations measured are close to the nominals the effect data are based on the nominal test concentrations.


The validity criteria were respected:

No mortality occurred in the control group over the test period.

The average number of juveniles per parent in the control was 63 after 22 days.


Normally, the No Observed Effect Concentration (NOEC) is determined as the concentration used in the study that is immediately below the Lowest Observed Effect Concentration (LOEC), the latter derived statistically from the data using the appropriate statistical test.


Reproduction and parental weight were found to be the most sensitive endpoints.

These data was found to be normally distributed and homogeneous. Using Dunnett’s and Bonferroni-t tests, the lowest Observed Effect Concentration (LOEC) was found to be 32 mg/L. Based on these statistical results the NOEC is 10 mg/L. An EC10and EC50for reproduction were determined as well they were 25.6 mg/L and 51.0 mg/L, respectively.

The EC50for parent survival was 148.6 mg/L.