Reaction mass of N-[3-(dimethylamino)propyl]docosanamide and N-[3-(dimethylamino)propyl]stearamide

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-07-11 to 2005-07-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]docosanamide

EC Number:

262-134-8

EC Name:

N-[3-(dimethylamino)propyl]docosanamide

Cas Number:

60270-33-9

Molecular formula:

C27H56N2O

IUPAC Name:

N-[3-(dimethylamino)propyl]docosanamide

Test material form:

solid

Details on test material:

- Name of test material: DIMAPDO lactate
- Molecular weight: 514.83 g/mol
- Analytical purity: 99.2 %
- Physical state: solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Two samples of control and each surviving test group were taken at 0 (fresh media), 24, 48, 72 (old and fresh media) and 96 hours (old media)
- Sample storage conditions before analysis: Further samples (in duplicate) were taken and stored at -20°C for further analysis, if necessary

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mg finely chopped test material were dispersed in dechlorinated tap water, with the aid of ultrasonication at 60°C for 2 hours, and the volume adjusted to 2 litres to give a 100 mg/L stock dispersion. These stock dispersions were then cooled to approximately 14°C prior to use. Aliquots (200, 360, 640, 1120 and 2000 mL) of the 100 mg/L stock dispersions were each separately dispersed in a final volume of 20 litres of dechlorinated tap water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L test concentrations respectively. Preliminary solubility tests showed that, in dechlorinated tap water, flocculation/precipitation of the test material occurred. To prevent this, a shielded propeller stirrer, set at a minimal speed to prevent foaming and/or bubble formation, was placed in each test vessel.
Each of the stock dispersions was inverted several times to ensure adequate mixing and homogeneity.
In order to saturate adsorptive active sites, the test vessels were pre-conditioned with the test concentration to be used in the test, approximately 24 hours prior to the test start and each media renewal.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Throughout the duration of the test the control was observed to be a clear, colourless solution whilst the 1.0 to 10 mg/L test concentrations were observed to be slightly cloudy, homogenous dispersions increasing in turbidity with increasing concentration.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Age at study initiation: juvenile
- Length at study end: mean standard length of 4.2 cm (SD = 0.1)
- Weight at study end: mean weight of 0.90 g (SD = 0.21)
- Feeding during test: no

ACCLIMATION
- Acclimation period: 13 d
- Acclimation conditions: same as test
- Type and amount of food: commercial trout pellets (discontinued approximately 24 hours prior to the start of the definitive test)
- Health during acclimation (any mortality observed): zero mortality in the 7 days prior to the start of the test

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

total hardness approx. 100 mg/L as CaCO3

Test temperature:

13.7°C - 16.0°C

pH:

7.3 - 8.3

Dissolved oxygen:

72% - 99% of Air Saturation Value
7.4 - 10.0 mg/L

Nominal and measured concentrations:

fresh media, untreated: measured test concentrations within 70% to 106% of nominal, with the exception of the 1.0 mg/L test concentration at 48 hours which showed a measured test concentration of 54% of nominal and the 5.6 mg/L test concentration at 72 hours which showed a measured test concentration of 136% of nominal
centrifuged samples: measured test concentrations within 21% to 119% of nominal
old media or expired test media, untreated: measured test concentrations within 48% to 115% of nominal
old media or expired test media, centrifuged: measured test concentrations within 10% to 88% of nominal
The majority of the concentrations with results outside the 80% to 120% confidence limits were below the No Observed Effect Concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 litre glass exposure vessels
- Type: closed
- Aeration: via propeller stirrers placed in tanks
- Renewal rate of test solution: daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.32 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated and partly softened laboratory tap water (water supply zone ZDB13 Alvaston)
- Total organic carbon: 1.220 to 1.99 mg/L
- Pesticides: 0 µg/L
- Total Chlorine: 0.0010 to 0.390 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours darkness with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED: mortality and sub-lethal effects were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: 0.1, 1.0,10, 100 mg/L
- Test concentrations: 1.0, 1.8, 3.2, 5.6, 10 mg/L
- Results used to determine the conditions for the definitive study: no mortalities at 0.10 and 1.0 mg/L, mortalities were observed at 10 and 100 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 5.6-10 mg/L

Effect concentrations 2

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

centrifuged

Basis for effect:

mortality (fish)

Remarks on result:

other: 2.6-6.6 mg/L

Effect concentrations 3

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

6.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

untreated

Basis for effect:

mortality (fish)

Remarks on result:

other: 4.8-9.1 mg/L

Effect concentrations 4

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.45

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

(DIMAPDO) untreated

Basis for effect:

mortality (fish)

Remarks on result:

other: 3.96 to 7.51 mg/L

Details on results:

- Other biological observations: Sub-lethal effects of exposure were observed at a test concentration of 10 mg/L. This response was the presence ofmoribund fish. After 20 and 22 hours exposure one fish was observed to be dead at each time point. These fish were removed from the test vessel and classed as mortalities for the 24-Hour time point. After 22.5 hours exposure two out of five fish were observed to be moribund. Due to the approach of the substantial severity limit (Animals (Scientific Procedures) Act 1986) these fish were killed and classed as mortalities for the 24-Hour time point. After 24 hours exposure the three remaining fish were observed to be moribund. These fish were killed and classed as mortalities for the 48-Hour time point.
- Mortality of control: 0%
- Other adverse effects control: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Throughout the duration of the preliminary test the control was observed to be a clear, colourless solution whilst the 1.0 to 10 mg/L test concentrations were observed to be slightly cloudy, homogenous dispersions increasing in turbidity with increasing concentration.

Reported statistics and error estimates:

An estimate of the LC50 values at 3 and 6 hours was given by inspection of the mortality data.
The LC50 value and associated confidence limits at 24 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al. 1977) using the ToxCalc computer software package (ToxCalc 1999) and at 48, 72 and 96 hours using the geometric mean method.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Analysis of mortality data:

 

Time (h)	LC50(95 % confidence limits)nominal concentrations(mg/L)	LC50(95 % confidence limits) mean measured concentrations untreated(mg/L)	LC50(95 % confidence limits) mean measured concentrations centrifuged (mg/L)
3	> 10	> 9.1	> 6.6
6	> 10	> 9.1	> 6.6
24	9.3 (6.7-13)	8.4 (5.8-12)	5.9 (3.4-10)
48	7.5 (5.6-10*)	6.6 (4.8-9.1*)	4.1 (2.6-6.6*)
72	7.5 (5.6-10*)	6.6 (4.8-9.1*)	4.1 (2.6-6.6*)
96	7.5 (5.6-10*)	6.6 (4.8-9.1*)	4.1 (2.6-6.6*)

* Concentrations resulting in 0% and 100% mortalities respectively

NOEC (nominal concentrations): 5.6 mg/L

NOEC (mean measured concentrations untreated media): 2.6 mg/L

NOEC (mean measured concentrations centrifugede media): 4.8 mg/L

Observations on the test animals

As the test material was observed to form a dispersion in the test diluent, microscopic inspection was performed on the gill filaments of the dead fish observed during the test. This examination revealed test material had adhered to the gill filaments indicating physical toxicity may have been a factor.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 based on nominal test concentrations was 7.5 mg/L with 95% confidence limits of 5.6 - 10 mg/L.
The 96-hour LC50 based on the time-weighted mean measured test concentrations of the centrifuged test media was 4.1 mg/L with 95% confidence limits of 2.6 - 6.6 mg/L.
The 96-hour LC50 based on the time-weighted mean measured test concentrations of the untreated test media was 6.6 mg/L with 95% confidence limits of 4.8 - 9.1 mg/L.
The results based on the time-weighted mean measured test concentrations of the centrifuged test media showed the results based on the soluble test material within the test system, whilst the time-weighted mean measured concentrations of the untreated test media showed the results based on the total amount of test material, both soluble and dispersed within the test system.

Executive summary:

In a 96-h acute toxicity study according toOECD guideline 203 (Fish, Acute Toxicity Test) 1992 and EU Method C.1 (Acute Toxicity for Fish) 1992, juvenile rainbow trouts (Oncorhynchus mykiss) were exposed to DIMAPDO lactate at nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg test substance/L (mean measured concentrations of untreated test medium: 0.63, 1.5, 2.9, 4.8 and 9.1 mg/L; mean measured concentrations of centrifuged test medium: 0.32, 0,76, 1.2, 2.6 and 6.6 mg/L) under semi-static conditions. The 96 -hour LC50 based on nominal test concentrations was 7.5 mg/L with 95% confidence limits of 5.6 - 10 mg/L. The 96 -hour LC50 based on mean measured concentrations of untreated test medium was 6.6 mg/L with 95% confidence limits of 4.8 - 9.1 mg/L.

The analysed test concentrations of the untreated solution (analysis of dispersed + dissolved test substance) and not of the centrifuged solution (analysis only of dissolved test substance) were chosen for further risk assessment because in the OECD guideline 201 study there was a discontinuity in the analysis of the concentrations of the centrifuged test solution and therefore the calculated values for the centrifuged test media were not valid. Additionally the examination of the dead fish revealed that the dispersed test material had adhered to the gill filaments indicating physical toxicity of the dispersed test material particles may be a factor in toxicity to fish.

Because the study was conducted with the lactic acid salt of DIMAPDO, the 96-h LC50 of DIMAPDO based on mortality and sublethal effects and the concentration analysis of the untreated test solution were calculated as 5.45 mg/L active ingredient.

Sub-lethal effects (moribundity) of exposure were observed at a nominal test concentration of 10 mg/L. Due to the approach of substantial severity limit these fish were killed and classed as mortalities at 24 and 48-hour time points respectively.

This toxicity study is classified as acceptable and satisfies the guideline requirement for a fish short-term toxicity study.

 

Results Synopsis

Test organism size/age: juvenile rainbow trouts (Oncorhynchus mykiss); mean length: 4.2 cm (SD = 0.1); weight: 0.90 g (SD = 0.21)

Test Type: semi-static (daily renewal)

 

LC50: 5.45 mg a.i./L                        95% C.I.: 3.96 to 7.51 mg a.i./L

NOEC:  3.96 mg a.i./L                           

Endpoint(s) Effected: mortality