N-octylpyridin-4-amine

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 05 April 2018, Experimental completion date 11 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-octylpyridin-4-amine

EC Number:

265-019-0

EC Name:

N-octylpyridin-4-amine

Cas Number:

64690-19-3

Molecular formula:

C13H22N2

IUPAC Name:

N-octylpyridin-4-amine

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each 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. Additional samples of each test concentration containing no algal cells were prepared at the start of the test and incubated alongside to provide samples for uninoculated analysis at 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

Definitive Test
Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 0.010, 0.032, 0.10, 0.32 and 1.0% v/v saturated solution.

Experimental Preparation
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0, 0.32, 0.10, 0.032 and 0.010% v/v saturated solution.
An aliquot (500 mL) of each of the 0.010, 0.032, 0.10, 0.32 and 1.0% v/v saturated solution stock solutions was separately inoculated with 2.9 mL of algal suspension to give the required test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0% v/v saturated solution.
The stock solutions and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis by Residue Analytical Services at Envigo - Eye

Test organisms

Test organisms (species):

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

Details on test organisms:

Test System and Supporting Information
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 kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±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 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 104 to 105 cells/mL.
A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item. The positive control was conducted between 06 November 2017 and 09 November 2017.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 22, 45 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
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.

Test conditions

Details on test conditions:

Exposure Conditions
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 group 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 8.58 x 105 cells per mL. Inoculation of 500 mL of test medium with 2.9 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 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 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Water Quality Criteria
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.

Results and discussion

Effect concentrations

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.0016 to 0.22 mg/L. A concentration dependent decline in measured test concentrations was observed at 72 hours in the range of less than the LOD of the analytical method employed (determined to be 0.00020 mg/L) to 0.18 mg/L.
Analysis of additional test samples incubated alongside the test which contained no algal cells showed measured test concentrations to range from 0.0016 to 0.21 mg/l (97% to 113% of the 0-Hour measured test concentrations) indicating that the decline in measured concentrations observed in those samples where algae were present was due to adsorption of the test item to the algal cells present rather than instability of the test item under the conditions of the test.
It was therefore considered appropriate to calculate the results based on the 0-Hour measured test concentrations only as these values give an indication of the concentration of test item to which the algae were exposed.


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 based on the 0-Hour measured test concentrations:

Inhibition of Growth Rate
ErC10 (0 to 72 hour): 0.014 mg/L
ErC20 (0 to 72 hour): 0.018 mg/L
ErC50 (0 to 72 hour): 0.027 mg/L; 95% confidence limits 0.024 to 0.031 mg/L
Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations 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, 0.0016 and 0.0070 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.0070 mg/L. Correspondingly the LOEC based on growth rate was 0.020 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 0.0048 mg/L
EyC20 (0 to 72 hour): 0.0067 mg/L
EyC50 (0 to 72 hour): 0.012 mg/L; 95% confidence limits 0.010 to 0.013 mg/L
Where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences between the control and 0.0016 mg/L test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 0.0016 mg/L. Correspondingly the LOEC based on yield was 0.0070 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 302 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.
Nominal cell density of control at 0 hours : 5.00 x 103 cells per mL
Mean cell density of control at 72 hours : 1.51 x 106 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 16% 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 to 72 hours) was 1% 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 0.0016 and 0.0070 mg/L, however misshaped cells were observed to be present in the 0.020 mg/L test cultures and cell debris was observed to be present in the test cultures at 0.056 and 0.22 mg/L 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 8.8 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.

Observations on Test Item Solubility
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, 0.0016 and 0.0070 mg/L test cultures were observed to be green dispersions. The 0.020 mg/L test cultures were observed to be pale green dispersions whilst the 0.056 and 0.22 mg/L test cultures were observed to remain 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 based on the 0-Hour measured test concentrations gave the following results:
Growth rate
EC50 = 0.027 mg/L
NOEC = 0.0070 mg/L
LOEC = 0.020 mg/L