Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with N1,N1-dimethyl-1,3-propanediamine

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:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A reliable/specific analytical method could not be found for this substance in water. Additionally, the test solutions were loaded from a 10 mg/L loading rate WAF at levels above the water solubility limit (< 0.9 mg/L at 20 degrees C according to Method A6; model predicted water solubility of 7E-10 mg/L at 25 C); hence the exposure concentrations in the algal study likely overloaded the carrying capacity of the water resulting in undissolved materials that may have exerted a physical effect on the organisms and confounded the reliability of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis[3-(dimethylamino)propyl]-9-nonyl-10-octylnonadecanediamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-[(2E)-oct-2-en-1-yl]-4-pentylcyclohexyl]nonanamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-octyl-4-pentylcyclohexyl]nonanamide

EC Number:

937-688-5

Cas Number:

1391530-05-4

Molecular formula:

C46H82N4O2 to C46H94N4O2

IUPAC Name:

N,N'-bis[3-(dimethylamino)propyl]-9-nonyl-10-octylnonadecanediamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-[(2E)-oct-2-en-1-yl]-4-pentylcyclohexyl]nonanamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-octyl-4-pentylcyclohexyl]nonanamide

Details on test material:

- Physical state: amber coloured viscous liquid
- Expiration date of the lot/batch: May 10, 2011
- Water solubility (under test conditions): Less than 0.9 mg/L at 20 degrees C according to Method A6; model predicted water solubility of 7E-10 mg/L at 25 degrees C according to Harlan report

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

1) OECD 2000. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures

A determination of the General PhysicoChemical Properties on the test material gave a water solubility value of less than 0.9 mg/L at 20 degrees C according to Method A6 and a water solubility of 7E-10 at 25 degrees C according to calculation methods.

Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organ carbon as an indicator of soluble organic substances in the WAF. Four WAFs of a nominal loading rate of 100 mg/L were prepared in reconstituted water. One loading rate was stirred for a period of 23 hours, one for a period of 47 hours, one for a period of 71 hours and another for a period of 95 hours. Pre-study work indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours.

Based on the results of the range finding tests the following loading rates were assigned to the definitive test: 0.01, 0.032, 0.1, 0.32 and 1.0 mg/L. Due to the need to test at relatively low test concentrations it was considered appropriate to prepare a 10 mg/L loading rate WAF from which serial dilutions were made to give the required test concentrations.

An amount of test item (100 mg) was added to the surface of 10 litres of culture medium to give the 10 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the undissolved material by filtering through a glass wool plug. The WAF was removed by mid depth siphoning (the first 100 mL discarded) to give the 10 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed there to be no microdispersions of test item present. A series of dilutions were made from the 10 mg/L loading rate WAF to give stock solutions of 1.0, 0.32, 0.10, 0.032 and 0.01 mg/L loading rate WAFs. An aliquot of each of the stock solutions was separately inoculated with algal suspension to give the required test concentrations of 0.01, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAF.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Observations on the test media were carried out during the mixing and testing of the WAFs. At the start of mixing the 10 mg/L loading rate WAF was observed to be a clear colourless media column with test item at the surface. Following the 23 hour stirring period and 1 hour standing period the WAF was observed to have formed a slightly cloudy media column. Microscopic examination of the WAF showed there to be microdispersions of test item present and hence the WAF was removed by filtration through a glass wool plug. After filtration no microdispersions of test item were observed to be present. AT the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72 hour test period all control and loading rate WAF test cultures were observed to be increasing pale green dispersions with increasing loading rate.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: Desmodesmus subspicatus, strain CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Method of cultivation: Cultures were maintained in the laboratory at a temperature of 21 degrees C under constant aeration and constant illumination. Prior to the start of the test sufficient master culture was added to culture media contained in conical flasks to give an initial cell density of 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 rpm) and constant illumination until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

no data

Test temperature:

Temperature was maintained at 24C throughout the test

pH:

The pH values of the control cultures were observed to increase from pH 7.4 to 7.7 at 0 hours to pH 7.7 to 7.9 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

Nominal loading rates were assigned to the definitive test at 0.01, 0.032, 0.1, 0.32 and 1.0 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
As in the range finding tests 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and three flasks each containing 100 ml were used for each treatment group. The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.75E5 cells per ml. Inoculation of test medium with the algal suspension gave an initial nominal cell density of 4E3 cells per ml and had not significant dilution effect on the final test concentration. The flasks were plugged with polyurethane foam bungs and incubated at 24 degrees C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380-730 nm) and constantly shaken at approximately 150 rpm for 72 hours. Samples were taken at 0, 24, 47 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.



GROWTH MEDIUM
The culture medium used for the range finding and definitive tests was the same as that used to maintain the stock culture. Stock solutions of the culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5.

- Standard medium used: yes

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate as the reference material at 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/L. Exposure conditions +data evaluation for the positive control were similar to the definitive test. Results from the positive control with po

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.23 - 0.3 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
Observation of abnormalities (for algal test):
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control of test cultures at 72 hours.

Any other information on results incl. tables

Table. Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)	Growth Rate (cells/ml/hour)		Yield (cells/mL)
	0 to 72 hours	% Inhibition	0 to 72 hours	% Inhibition
Control R1 R2 R3 R4 R5 R6 Mean SD	0.053 0.053 0.054 0.053 0.053 0.053 0.053 0.000	--	1.73E5 1.71E5 1.86E5 1.82E5 1.77E5 1.76E5 1.77E5 5.53E3	--
0.01 R1 R2 R3 Mean SD	0.060 0.059 0.051 0.057 0.005	[13] [11] 4 [7]	4.40E5 2.68E5 3.97E5 2.77E5 4.72E5	[35]
0.032 R1 R2 R3 Mean SD	0.064 0.061 0.064 0.063 0.002	[21] [15] [21] [19]	4.01E5 3.25E5 3.91E5 3.72E5 4.11E4	[110]
0.1 R1 R2 R3 Mean SD	0.057 0.054 0.056 0.056 0.002	[8] [2] [6] [5]	2.33E5 1.98E5 2.14E5 2.15E5 1.77E4	[21]
0.32 R1 R2 R3 Mean SD	0.038 0.041 0.037 0.039 0.002	28 23 30 27	5.92E4 7.18E4 5.29E4 6.13E4 9.65E3	65
1.0 R1 R2 R3 Mean SD	0.015 0.015 0.013 0.014 0.001	72 72 75 73	7.66E3 7.65E3 6.08E3 7.13E3 9.08E2	96

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Desmodesmus subspicatus was investigated over a 72 hour period and under the conditions of the study gave the following results: EL50 growth rate 0.58 mg/L; EL50 yield 0.26 mg/L; NOEL for both growth and yield of 0.1 mg/L.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green algaDesmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No. 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

 

Methods:

A determination of the General Physico-Chemical Properties study conducted on the test material gave a water solubility value for the test material of less than 0.9 mg/L at 20 degrees C according to Method A6 and using calculation methods the water solubility estimate at 25 degrees C is 7E-10 mg/L. Despite extensive method development work a reliable and specific analytical method could not be found for the test item in water. Even Total organ carbon analysis was attempted however the measured test concentrations were found to be less than the limit of quantitation of 1 mg C/L.

 

Following preliminary range-finding tests, Desmodesmus subspicatuswas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.01, 0.032, 0.1, 0.32 and 1.0 mg/L (three replicate flasks per concentration0 for 72 hours, under constant illumination and shaking at a temperature of 24 degrees C. Samples of the algal population were removed daily and cell concentrations determined for each control and treatment group using a Coulter Multisizer Particle Counter.

 

 

Results:

The effect of the test item on the growth ofDesmodesmus subspicatuswas investigated over a 72 hour period and under the conditions of the study gave the following results: EL50 growth rate 0.58 mg/L; EL50 yield 0.26 mg/L; NOEL for both growth and yield of 0.1 mg/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates.