Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride

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:

Experimental start date 03 October 2016 Experimental completion date 25 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride

EC Number:

947-079-6

IUPAC Name:

Reaction mass of N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethyldocosan-1-aminium chloride and N-(3-{[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]amino}propyl)-N,N-dimethylicosan-1-aminium chloride

Test material form:

other: paste

Specific details on test material used for the study:

Identification: Methoxycinnamidopropyl Behendimonium Chloride
Physical state/Appearance: Off-white paste
Batch: INV-1603010
Purity: 29.37%
Expiry Date: 01 February 2017
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range Finding Test
A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Samples were taken from the control and each loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Validation of Mixing Period
Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of dissolved test item in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionized reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by filtration through a glass wool plug and Postlip filter paper and samples taken for chemical analysis.
It is evident from this work that increasing the stirring period did not significantly increase the amount of dissolved test item in the WAF and so preparation of the WAF was maintained at 24 hours.

Range-Finding Tests
Nominal amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. 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 mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded). The WAFs were then passed through two Postlip filter papers to remove as much undissolved test item as possible. After filtration the WAFs had a slight opalescent appearance though microscopic examination showed no undissolved test item to be visible.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (5.4 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.

A nominal amount of test item (20 mg) was weighed onto a glass slide and suspended within 20 liters of culture medium to give the 1.0 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. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further concentrations of 0.10, 0.010 and 0.0010 mg/L loading rate WAF.
An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.1 mL) to give the required test concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/L loading rate WAF.

Definitive Test
A nominal amount of test item (20 mg) was weighed onto a glass slide and suspended within 20 liters of culture medium to give the 1.0 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. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further concentrations of 0.32, 0.10, 0.032 and 0.010 mg/L loading rate WAF.
An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (8.2 mL) to give the required test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAF

Test organisms

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 – 10^5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

The pH values in the definitive test was observed to increase from pH 7.6 at 0 hours to pH 8.4 at 72 hours.
The pH value of the control cultures was observed to increase from pH 7.5 at 0 hours to pH 8.3 at 72 hours.

Nominal and measured concentrations:

Range finding test
Nominal concentrations of 0.0010, 0.010, 0.10, 1.0 mg/L loading rate WAF
Chemical analysis of the 0.010, 0.10 and 1.0 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations to range from 0.10 to 0.46 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of 0.048 to 0.067 mg/L indicating that the test item was unstable over the test duration.

Definitive test
Nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAD
Analysis of the test preparations at 0 hours showed measured concentrations to range from less than the limit of quantification (LOQ) of the analytical method, determined to be 0.0093 mg/L, to 0.71 mg/L. A decline in measured concentrations was observed at 72 hours in the range of less than the LOQ to 0.020 mg/L indicating that the test item was unstable over the duration of the test.

Details on test conditions:

Range-Finding Tests
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a haemocytometer and light microscope. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC 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.
After 72 hours the cell density of each flask was determined using a haemocytometer and light microscope.
It was not possible to monitor algal growth using a Coulter® Multisizer Particle Counter due to interference from the undissolved test item.
The results of the initial range-finding test showed significant inhibition of growth occurred at both 10 and 100 mg/L loading rate WAF and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.0010, 0.010, 0.10 and 1.0 mg/L for a period of 72 hours. Due to the need to test at low test concentrations, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give the remaining test concentrations.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC 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.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Due to the need to test at low test concentrations, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give the remaining test concentrations.
Exposure Conditions
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 2.74 x 10^5 cells per mL. Inoculation of 450 mL of test medium with 8.2 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °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.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.43 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.032 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.1 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.08 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.032 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

0.1 other: mg/L Loading Rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Range-finding Tests
The results showed no effect on growth at 0.0010 and 0.010 mg/L loading rate WAF. However, growth was observed to be reduced at 0.10, 1.0, 10 and 100 mg/L loading rate WAF.
Based on this information loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were selected for the definitive test.

Definitive Test
Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Growth Data
It is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL10 (0 - 72 h) : 0.044 mg/L loading rate WAF
ErL20 (0 - 72 h) : 0.10 mg/L loading rate WAF
ErL50 (0 - 72 h) : 0.43 mg/L loading rate WAF*
Where ErLx is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all loading rates 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). There were no statistically significant differences between the control, 0.0010 and 0.032 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 0.032 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 0.10 mg/L loading rate WAF.
* It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.

Inhibition of Yield
EyL10 (0 - 72 h) : 0.036 mg/L loading rate WAF
EyL20 (0 - 72 h) : 0.048 mg/L loading rate WAF
EyL50 (0 - 72 h) : 0.080 mg/L loading rate WAF; 95% confidence limits 0.068 – 0.094 mg/L loading rate WAF
Where EyLx is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out. There were no statistically significant differences between the control, 0.010 and 0.032 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 0.032 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 0.10 mg/L loading rate WAF.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L
EyC50 (0 – 72 h) : 0.79 mg/L; 95% confidence limits 0.70 – 0.89 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 1.0 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 177 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.00 x 10^3 cells per mL

Mean cell density of control at 72 hours : 8.84 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 10% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.010, 0.032 and 0.10 mg/L loading rate WAF. The 0.32 mg/L loading rate WAF test cultures were observed to be clumped together with cell debris present, very few cells were observed to be present in the 1.0 mg/L loading rate WAF test cultures.

Water Quality Criteria

Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.5 at 0 hours to pH 8.3 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.

Vortex Depth Measurements

The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAFs.

At the start of mixing the 1.0 mg/L loading rate WAF was observed to have formed a clear colorless media column with test item adhered to the glass slide, some flakes of test item had dispersed within the media column. After stirring the WAF was observed to be a clear colorless media column with a few flakes of test item floating at the media surface and settled on the bottom of the vessel, no test item was visible on the glass slide. After a 1-Hour standing period the WAF was observed to be a clear colorless media column with a few flakes of test item floating settled on the bottom of the vessel. Microscopic examination of the WAF after siphoning showed there to be no undissolved test item present.

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.010 and 0.032 mg/L loading rate WAF test cultures were observed to be very pale green dispersions. The 0.10 mg/L loading rate WAF test cultures were extremely pale green dispersions whilst the 0.32 and 1.0 mg/L loading rate WAF test cultures were clear colorless solutions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:
Growth rate
EL50 (0 - 72 h) : 0.43 mg/L loading rate WAF
NOEL: 0.032 mg/L
LOEL : 0.10 mg/L

Yield
EL50 (0 - 72 h) : 0.080 mg/L loading rate WAF; 95% confidence limits 0.068 – 0.094 mg/L loading rate WAF
NOEL: 0.032 mg/L
LOEL : 0.10 mg/L

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with 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) 761/2009.

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured concentrations to range from less than the limit of quantification (LOQ) of the analytical method, determined to be 0.0093 mg/L, to 0.71 mg/L. A decline in measured concentrations was observed at 72 hours in the range of less than the LOQ to 0.020 mg/L indicating that the test item was unstable over the duration of the test.

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 only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Resonse Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	0.43	*	0.032	0.1
Yield	0.08	0.068 - 0.094	0.032	0.1

* It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.