Reaction mass of N,N’-bis-(3-methoxypropyl)-C22(branched)-alkyldiamide and N,N’-bis-(3-methoxypropyl)-C17(branched)-alkyldiamide

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 Starting Date: 8 May 2014; Experimental Completion Date: 25 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal 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,N’-bis-(3-methoxypropyl)-C22(branched)-alkyldiamide and N,N’-bis-(3-methoxypropyl)-C17(branched)-alkyldiamide

EC Number:

940-953-8

Molecular formula:

not applicable UVCB

IUPAC Name:

Reaction mass of N,N’-bis-(3-methoxypropyl)-C22(branched)-alkyldiamide and N,N’-bis-(3-methoxypropyl)-C17(branched)-alkyldiamide

Test material form:

other: solid block

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Definitive Test:
- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L loading rates

- Sampling method:
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.

- Sample storage conditions before 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:

CULTURE MEDIUM:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

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

VALIDATION OF MIXING PERIOD:
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations 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 siphon and samples taken for chemical analysis.

There was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

RANGE-FINDING TESTS:
The loading rates to be used in the definitive test were determined by preliminary range-finding tests.

The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours.

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. Prior to use the test item was heated to 80 °C in order to aid weighing.

Amounts of test item (20 and 200 mg) were each separately added to the surface of 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. 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 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (7.7 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.

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. All samples were stored frozen prior to analysis.

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 0.010, 0.10 and 1.0 mg/L for a period of 72 hours. Due to the need to test at relatively low loading rates, a single WAF at a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give the remaining test concentrations. Prior to use the test item was heated to 80 °C in order to aid weighing.

An amount of test item (20 mg) was added to the surface of 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. 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 WAF 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) to give the 1.0 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed there to be no micro-dispersions of test item present. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.10 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 (7.8 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.

Exposure conditions in the second range-finding test were the same as those in the initial test.

DEFINITIVE TEST:
Based on the results of the range-finding tests the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation:
Nominal amounts of test item (20, 64, 20, 64 and 200 mg) were each separately added to the surface of 20, 20, 2, 2 and 2 liters of culture medium to give the 1.0, 3.2, 10, 32 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. Microscopic 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) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test item present.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (5.7 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

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. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1°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 10E3 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 10E4 – 10E5 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 culture mediums pH was adjusted to 7.5 with 0.1N NaOH or HCl.

Nominal and measured concentrations:

Definitive Test:
Nominal: 1.0, 3.2, 10, 32, 100 mg/L loading rate WAF
Measured: See results section.

Details on test conditions:

DEFINITIVE TEST:
Exposure Conditions:
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 4.40 x 10E5 cells per mL. Inoculation of 500 mL of test medium with 5.7 mL of this algal suspension gave an initial nominal cell density of 5 x 10E3 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.


CULTURE MEDIUM
- Standard medium used: yes (as defined in OECD 201 Guideline).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Test Organism Observations:
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria:
The pH of the control and each test concentration was determined at initiation of the test and after 72 hours exposure. The temperature within the incubator was recorded daily.

Vortex Depth Measurements:
The vortex depth was recorded at the start and end of the mixing period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 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.:

32 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:

EL50

Effect conc.:

93 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 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:

LOELR

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

RANGE-FINDING TESTS:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding tests are given in Table 1 and Table 2 (attached background material).

The results showed no effect on growth at 0.010, 0.10 and 1.0 mg/L loading rate WAF. However, growth was observed to be reduced at 10 and 100 mg/L loading rate WAF.

Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Chemical analysis of the 10 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured test of 0.25 and 0.72 mg/L respectively were obtained whilst concentrations of 0.14 and 0.59 mg/L respectively were obtained at 72 hours. The decline in measured concentration observed at 72 hours was considered to be due to possible instability and/or adsorption of the test item to the algal cells present.

DEFINITIVE TEST:
Chemical Analysis of Test Loading Rates
Analysis of the test preparations at 0 hours (see Appendix 'Analytical Investigations') showed measured test concentrations to range from 0.030 to 0.28 mg/L. A decline in measured test concentration was observed at 72 hours in the range of 0.0094 to 0.19 mg/L (24% to 69% of the 0-Hour measured test concentrations).

The dissolved test item may have been one or several components of the test item. 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:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3. Daily specific growth rates for the control cultures are given in Table 4. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 5.
See attached background material for Tables 3, 4 and 5.

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) : 88 mg/L loading rate WAF
ErL20 (0 - 72 h) : 97 mg/L loading rate WAF
ErL50 (0 - 72 h) : >100 mg/L loading rate WAF

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, 1.0, 3.2, 10 and 32 mg/L loading rate WAFs (P≥0.05), however the 100 mg/L loading rate was significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 32 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 100 mg/L loading rate WAF.

Inhibition of Yield
EyL10 (0 - 72 h) : 75 mg/L loading rate WAF
EyL20 (0 - 72 h) : 81 mg/L loading rate WAF
EyL50 (0 - 72 h) : 93 mg/L loading rate WAF

There were no statistically significant differences between the control, 1.0, 3.2, 10 and 32 mg/L loading rate WAFs (P≥0.05), however the 100 mg/L loading rate was significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 32 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 100 mg/L loading rate WAF.

VALIDATION CRITERIA:
The following data show that the cell concentration of the control cultures increased by a factor of 119 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: 3.68 x 10E3 cells per mL
Mean cell density of control at 72 hours: 4.38 x 10E5 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 1.0, 3.2, 10 and 32 mg/L loading rate WAF, however swollen cells were observed to be present in the test cultures at 100 mg/L loading rate WAF.

WATER QUALTIY CRITERIA:
The pH values of the control and each test concentration are given in Table 3 (attached background material). Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures (see Table 3) was observed to increase from pH 7.4 at 0 hours to pH 7.6 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.

VOTEXT 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 both the start and end of the mixing period, and following a 1-Hour standing period, all loading rate WAFs were observed to have formed clear colorless media columns with test item floating at the surface. Microscopic examination of the WAFs showed there to be micro-dispersions of test item present and hence it was considered justifiable to filter the aqueous phase through a glass wool plug. Microscopic examination of the WAFs after filtration showed there to be no micro-dispersions of 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, 1.0, 3.2, 10 and 32 mg/L loading rate WAF test cultures were observed to be green dispersions whilst the 100 mg/L loading rate WAF test cultures were observed to be pale green dispersions.

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): 1.1 mg/L; 95% confidence limits 0.91 – 1.2 mg/L
EyC50 (0 – 72 h: 0.51 mg/L; 95% confidence limits 0.45 – 0.59 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

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

Reported statistics and error estimates:

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) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

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 >100 mg/L loading rate WAF
NOEC: 32 mg/L loading rate WAF
LOEC: 100 mg/L loading rate WAF

Yield;
EL50: 93 mg/L loading rate WAF
NOEC: 32 mg/L loading rate WAF
LOEC: 100 mg/L loading rate WAF

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 1.0, 3.2, 10, 32 and 100 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 test concentrations to range from 0.030 to 0.28 mg/L. A decline in measured test concentration was observed at 72 hours in the range of 0.0094 to 0.19 mg/L (24% to 69% of the 0-Hour measured test concentrations). 

 

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:

 

Response Variable	EL50 (mg/L Loading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	>100	32	100
Yield	93	32	100