N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 21, 2003 to February 26, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Measurement of test concentrations:
- Pre-study stability analysis showed the test substance to be unstable in the test medium. Therefore in order to maintain concentrations of parent test substance as high as possible, the test solutions were renewed daily. Samples of freshly prepared and 24 h old test media were taken for analysis throughout the test.

- Chemical analysis of the freshly prepared test samples showed the measured test concentrations to range from 83% to 120% of the nominal test concentrations for the majority of samples taken. One low value of 78 % of nominal and six high values in the range of 128 % to 193 % of nominal were determined. Where these analyses showed low or high values, frozen duplicate samples were analysed. However, the results of these analyses were inconclusive. It was considered that the test substance had degraded or adsorbed to the glassware under the conditions of storage, and during thawing given that the majority of the samples stored frozen showed a marked decline in the measured test concentrations when compared to the original analyses.

- Analysis of the old or expired test samples showed a marked decline in the measured test concentrations which were shown to range from less than the Limit of Quantitation (LOQ) of the analytical method employed to 59 % of nominal. This decline was considered to be due to the unstable nature or adsorptive behaviour of the test substance in the test diluent and adsorption to the algal cells used as feed for the daphnids during the test, as indicated by the pre-test stability analyses.

- Given the marked decline in the measured test concentrations it was considered appropriate to calculate the results of the definitive test based on the time-weighted mean measured test concentrations in order to show “worst case” analysis of the data.

Vehicle:

no

Details on test solutions:

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary rangefinding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/l. The test material was dispersed directly in water.
An amount of test material (100 mg) was dispersed in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock dispersion. A dilution was made from this to give the test concentration of 10 mg/l. Aliquots (10, 32, 100 and 320 ml) of the 10 mg/l test concentration were each separately dispersed in a fmal volume of 1 litre of reconstituted water to give the 0.10, 0.32, 1.0 and 3.2 mg/l test concentrations respectively. To prepare the 0.010 and 0.032 mg/l test concentrations aliquots (10 and 32 ml) of the 1.0 mg/l test concentration were each separately dispersed in a final volume of 1 litre of reconstituted water. Visual inspection of the prepared test concentrations showed them to be clear, colourless solutions.
Prior to use the stock dispersion and each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 21°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test material.

Range-finding Test
No immobilisation was observed at the test concentrations of 0.010, 0.032 and 0.10 mg/L throughout the test. However, 100 % immobilisation was observed at 0.32, 1.0, 3.2 and 10 mg/L after 48 hours exposure.
Based on this information test concentrations of 0.0032, 0.010, 0.032, 0.10 and 0.32 mg/L were selected for the definitive test.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water at a temperature of approximately 21°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

in the range 242 to 260 mg/L as CaCO3

Test temperature:

21 C

pH:

7.9 +/- 0.7

Dissolved oxygen:

>7.8

Salinity:

Fresh water

Nominal and measured concentrations:

3.3.2.4 Verification of test concentrations
Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 1,5, 8, 12, 15 and 20 and of the expired (24-hour old) test preparations on Days 1, 2, 6, 9, 13, 16 and 21.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Nominal test concentrations: Control, 0.0032, 0.010, 0.032, 0.10 and 0.32 mg/L
Observed TWA concentrations: BDL, 0.0017, 0.0039, 0.024, 0.066 and 0.21 mg/L

Details on test conditions:

Exposure conditions
For each concentration a single daphnid was placed in 100 ml of the test preparation in 150 ml glass flasks which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The flasks were maintained at approximately 21°C with a photoperiod of 16 hours light (458 to 612 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. Each vessel was randomly assigned to a position in the laboratory. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test material.
Pre-study stability analysis showed the test material to be unstable in the test medium. Therefore in order to maintain concentrations of parent test material as high as possible, the test solutions were renewed daily. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.
Each daphnid received approximately 2.0 to 7.0 III of a unicellular algal culture (Chiorella sp.) daily. Feeding was at a level of approximately 0.1 - 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
On a daily basis the numbers of live and dead of the "Parental" (PI) generation, the numbers of live and dead "Filial" (FI) Daphnia and the number of discarded unhatched eggs were counted.
An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
The number of Daphnia with eggs or young in the brood pouch was detennined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
At the end of the test, the length of each surviving parent animal was detennined.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.024 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.066 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.034 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: 0.026 - 0.045 mg/L

Details on results:

Exposure of Daphnia magna to the test substance resulted in significant mortalities at the test concentrations of 0.066 and 0.21 mg/L (based on time-weighted mean measured test concentrations) leading to 100 % mortalities by days 18 and 3, respectively.
- The 14 and 21 d EC50 (immobilisation) values for the parental Daphnia generation (P1) were calculated to be 0.095 mg/L and 0.034 mg/L (based on the time-weighted mean measured test concentrations) with 95% confidence limits of 0.072 —0.12 mg/L and 0.026 - 0.045 mg/L.
- The 21 d NOEC and LOEC were determined to be 0.066 and 0.024 mg/L (based on the time-weighted mean measured test concentrations) respectively.

Results with reference substance (positive control):

NA

Reported statistics and error estimates:

Evaluation of data
The EC50 (immobilisation) value at 24 hours was estimated by inspection of the data.
The EC50 (immobilisation) values and associated confidence limits at 48 hours, 14 and 21 days were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999) and at 96 hours and 7 days by the geometric mean method as follows:
EC50 value = SQRT(C1 X C2)
Where:
C1 = c showing 0% immobilisation
C2 = c showing 100% immobilisation

When only one partial response is shown the trimmed Spearman-Karber method is appropriate.
The EC50 (reproduction) value and associated confidence limits after 21 days were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
For the estimation of the "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration (NOEC) the numbers of live young produced per adult over the duration of the test for the control, 0.0032, 0.010 and 0.032 mg/l test groups were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) (see Appendix 3). Results from the control, 0.0032, 0.010 and 0.032 mg/l test groups Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Toxicity of the test substance to Daphnids followed a positive dose response for immobility and reproduction. There were no deaths among the control Daphnia in this study, meeting required validation criterion of ≤20 %.The mean number of live offspring per surviving adult was 80 for the control group, meeting the validation criterion of ≥60.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 21 days NOEC and LOEC of the test substance for effects on reproduction and survival rates of Daphnia magna were 0.024 and 0.066 mg/L (TWA measured concentration), respectively.

Executive summary:

A long-term study was conducted to determine the reproductive toxicity of the test substance to water flea (Daphnia magna) according to the OECD Guideline 211 and EU Method C.20, in compliance with GLP. Daphnia were exposed for 21 d under semi-static conditions (daily renewal). Analytical dose verification was conducted. Given the marked decline in the measured test concentrations it was considered appropriate to calculate the results of the definitive test based on the time-weighted average (TWA) measured test concentrations in order to show “worst case” analysis of the data. Exposure resulted in significant mortality at the test concentrations of 0.066 and 0.21 mg/L (TWA measured concentration), leading to 100 % mortality by Days 18 and 3, respectively. The 14 and 21 d EC50 (immobilisation) values for the parental Daphnia generation (P1) were calculated to be 0.095 and 0.034 mg/L (TWA measured concentration), with 95 % confidence limits of 0.072 —0.12 mg/L and 0.026 - 0.045 mg/L. Further, the 21 days NOEC and the LOEC were determined to be 0.024 and 0.066 mg/L (TWA measured concentration), respectively. Under the study conditions, the 21 days NOEC and LOEC of the test substance for effects on reproduction and survival rates of Daphnia magna were 0.024 and 0.066 mg/L (TWA measured concentration), respectively (Wetton, 2004).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.024 mg/L

Additional information

A long-term study was conducted to determine the reproductive toxicity of the test substance to water flea (Daphnia magna) according to the OECD Guideline 211 and EU Method C.20, in compliance with GLP. Daphnia were exposed for 21 d under semi-static conditions (daily renewal). Analytical dose verification was conducted. Given the marked decline in the measured test concentrations it was considered appropriate to calculate the results of the definitive test based on the time-weighted average (TWA) measured test concentrations in order to show “worst case” analysis of the data. Exposure resulted in significant mortality at the test concentrations of 0.066 and 0.21 mg/L (TWA measured concentration), leading to 100 % mortality by Days 18 and 3, respectively. The 14 and 21 d EC50 (immobilisation) values for the parental Daphnia generation (P1) were calculated to be 0.095 and 0.034 mg/L (TWA measured concentration), with 95 % confidence limits of 0.072 —0.12 mg/L and 0.026 - 0.045 mg/L. Further, the 21 days NOEC and the LOEC were determined to be 0.024 and 0.066 mg/L (TWA measured concentration), respectively. Under the study conditions, the 21 days NOEC and LOEC of the test substance for effects on reproduction and survival rates of Daphnia magna were 0.024 and 0.066 mg/L (TWA measured concentration), respectively (Wetton, 2004).