N,N'-bis{3-[di-(branched and cyclic) alkylamino]alkyl}alkanediamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-11-20 to 2015-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

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.
Samples were frozen prior to analysis.
Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Vehicle:

no

Details on test solutions:

Range-finding test

Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give 10 and 100 mg/L loading rates respectively. After the addition of the test item the water 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 rates. Microscopic observations of the WAFs showed there to be no micro-dispersions or undissolved test item present.
An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (6.1 mL) to give the required test concentrations of 10 and 100 mg/L.

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.
The test samples were prepared as above using 20 mg of the test material in 20 L culture medium to give the 1.0 mg/L loading rate. A series of dilutions were made from the 1.0 mg/L stock solution to give further stock solutions of 0.10 and 0.010 mg/L.
An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (9.2 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L.

Initial experiment
Based on the range finders nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were asssigned to the initial study..
The test samples were prepared as above using 20, 32, 20, 64 and 200 mg of the test material in 20, 10, 2.0, 2.0 and 2.0 L culture medium respectively to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates.
An aliquot (1L) of each of theloading rate WAFs was separately inoculated with algal suspension (4.7 mL) to give the required test concentrations of1.0, 3.2, 10, 32 and 100 mg/L.

Definitive test
The test sample was prepared as above using 20 mg of the test material in 20L culture medium to give a 1.0 mg/L loading rate. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.32 and 0.10 mg/L laoding rate WAF.
An aliquot (1L) of each of the stock solutions was separately inoculated with 5.2 mL of algal suspension to give the required test concentrations of 0.10, 0.32, and 1.0 mg/L loading rate WAF.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): 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 constant illumination at 21 ± 1 °C.
ACCLIMATION
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 10E03 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 10E04 - 10E05 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not specified

Test temperature:

24 ± 1 ºC

pH:

8.6 - 8.7

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations
1st Range finder: 10 and 100 mg/L loading rates
2nd Range finder: 0.010, 0.10 and 1.0 mg/L loading rates
Initial test: 1.0, 3.2, 10, 32 and 100 mg/L loading rates
Definitive test: 0.10, 0.32, and 1.0 mg/L loading rates

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 used 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 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 illuminationg (intensity approximately 700 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
The results of the initial range-finding test showed significant inhibition of growth occurred at both the 10 and 100 mg/L loading rates and so a second range-finding test was conducted. Exposure conditions in the second range-finding test were the same as those in the initial test.
Initial and Definitive test
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution 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 9.62E05 cells per mL. Inoculation of 1L of test medium with 5.2 mL of this algal suspension gave an initial nominal cell density of 5E03 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):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate WAF

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate WAF

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: loading rate WAF

Details on results:

Chemical analysis of loading rates
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less that the LOQ (0.00090mg/L). This does not infer that there was no test item in solution, just that any dissolved test item was at a concentration of less than the LOQ.
Given thattoxicity 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
(see attached tables)
The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-hour exposure period.
ErL10 (0-72 h) > 1.0 mg/L loading rate WAF
ErL20 (0-72 h) > 1.0 mg/L loading rate WAF
ErL50 (0-72 h) > 1.0 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 rate WAF test groups using one-way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control. There were no statistically significant differences between the control, 0.10, 0.32 and 1.0 mg/L loading rate WAF (P>= 0.05) and therefore the NOELR based on growth rate was 1.0 mg/L loading rate WAF.
Yield data
EyL10 (0-72 h) = 0.97 mg/L loading rate WAF (no 95% CI calculated as the data generated does not fit the models available)
EyL20 (0-72 h) > 1.0 mg/L loading rate WAF
EyL50 (0-72 h) > 1.0 mg/L loading rate WAF
Where EyLx is the loading rate that reduced yield by x%
Statistical analysis of the daat indicated no statistically significant differences between the control, 0.10, 0.32, and 1.0 mg/L loading rate WAFs (P.=0.05) and therefore the LOELR based on yield was 1.0 mg/L loading rate WAF.
Observation on culture
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.
Observations on test item solubility
At both the start and end of the mixing period, and following a 1-hour standing period, the 1.0 mg/L laodingrate WAF was observed to have formed a clear colourless media column with oily globules of test item floating at the surface. Microscopic examination of the WAFs showed there to be no micro-dispersions or globules of test item 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, 0.10, 0.32 and 1.0 mg/L l;aodingrate WAF test cultures were observed to be green dispersions.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

See results above

Validity criteria fulfilled:

yes

Conclusions:

Exposure of the test item to Pseudokirchneriella subcapitata gave EL50 values greater than the 1.0 mg/L loading rate WAF. The No Observed Effect Concentration was 1.0 mg/L loading rate WAF. The study showed that the test item was not toxic to algae at a concentration that is in excess of the water solubility of the substance.

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 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 761/2009.

Methods

Due to the low aqueous solubility and pure nature of the test item, for the purposes of the definitive test, the test medium was prepared as a slow stir saturated solution. Following preliminary range-finding tests and an initial test, Pseudokirchneriella subcapitata was exposed to WAFs of the test item at nominal loading rates of 0.01, 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

Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the LOQ (0.00090 mg/L) of the analytical method used. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less thanthe LOQ.

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

Exposure of the test item to Pseudokirchneriella subcapitata gave EL50 values greater than the 1.0 mg/L loading rate WAF. The No Observed Effect Concentration was 1.0 mg/L loading rate WAF.

The study showed that the test item was not toxic to algae at a concentration in excess of the water solubility of the substance.

Description of key information

Exposure of the test item to Pseudokirchneriella subcapitata gave EL50 values greater than the 1.0 mg/L loading rate WAF. The No Observed Effect Concentration was 1.0 mg/L loading rate WAF.

The study showed that the test item was not toxic to algae at a concentration in excess of the water solubility of the substance.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

The substance is a cationic surfactant type molecule with a low estimated CMC which accounts for the lack of ready biodegradation (16% in 28 days) observed. Algae, like sewage sludge micro-organisms, are anionic and therefore it is likely that in the WAF, these cationic molecules surround the relatively small number of algae that are in the solution, reducing their capacity to take up minerals and potentially even blocking active sites. Considering the results of the algal study, it appears that the critical concentration is somewhere >1 mg/L loading rate WAF.

In the environment it is unlikely that this situation would occur as the substance would be totally adsorbed to suspended solids, sediment and organic matter before it could reach a concentration effecting algal cells.

Although there would be a similar effect in the ASRI, with sewage sludge micro-organisms, the impact is mitigated due to hydrophobicity and therefore the effect is minor in comparison to that observed in the algal study.

As the 1.0 mg/L loading rate is much higher than the experimentally derived water solubility of the substance (less than 2.04E-06 g/L)the result of the study should not be used for determination of the PNEC, risk assessment or for the purpose of Classification and Labelling.

A key study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. 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 761/2009.

Due to the low aqueous solubility and pure nature of the test item, for the purposes of the definitive test, the test medium was prepared as a slow stir saturated solution. Following preliminary range-finding tests and an initial test, Pseudokirchneriella subcapitata was exposed to WAFs of the test item at nominal loading rates of 0.01, 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.

Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the LOQ (0.00090 mg/L) of the analytical method used. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less thanthe LOQ.

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

Exposure of the test item to Pseudokirchneriella subcapitata gave EL50 values greater than the 1.0 mg/L loading rate WAF. The No Observed Effect Concentration was 1.0 mg/L loading rate WAF.

The study showed that the test item was not toxic to algae at a concentration in excess of the water solubility of the substance.