Pyridine-2-carboxylic acid

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:

11 Aug - 24 Oct 2017

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

Pyridine-2-carboxylic acid

EC Number:

202-719-7

EC Name:

Pyridine-2-carboxylic acid

Cas Number:

98-98-6

Molecular formula:

C6H5NO2

IUPAC Name:

pyridine-2-carboxylic acid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 1.0, 3.2, 10, 32, 100 mg/L
- Sampling method: Samples were taken at the beginning (0 h) and the end of the study (72 h).
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted 1 L to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (1.9 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No, test solutions were clear and colorless.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): 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.
- Preparation of culture for testing: 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 10E+03 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 10E+04 - 10E+05 cells/mL.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

test start: 7.3 (control), 7.3 (1.0 mg/L), 7.3 (3.2 mg/L), 6.9 (10 mg/L), 5.2 (32 mg/L), 5.2 (100 mg/L)
test end: 7.7 (control), 7.9 (1.0 mg/L), 7.9 (3.2 mg/L), 7.7 (10 mg/L), 6.6 (32 mg/L), 5.6 (100 mg/L)

Nominal and measured concentrations:

nominal: control, 1.0, 3.2, 10, 32, 100 mg/L
measured (0 h): < LOQ, 0.875, 3.06, 9.58, 31.1, 101 mg/L
measured (72 h): < LOQ, 0.824, 3.04, 9.60, 31.6, 102 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: glass, 250 mL, headspace: 150 mL, fill volume: 100 mL
- Aeration: constantly shaken at approx. 150 rpm
- Initial cells density: 5E+03 cells/mL
- Control end cells density: 9.61E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1 N NaOH or HCl.
- Culture medium different from test medium: same as test
- Intervals of water quality measurement: The water quality parameters were checked regularly.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
- Other: The shape and size of the algal cells was inspected microscopically and any abnormalities recorded

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L (perfomed two times)
- Results used to determine the conditions for the definitive study: The results of the initial test showed a flat response occurred in terms of inhibition of growth rate. The results of the second test showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (tested: 28 Nov - 01 Dec 2016)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 11 - 16 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 5.6 - 7.1 mg/L

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 72 h there were no abnormalities detected in the control or test cultures at 1.0 and 3.2 mg/L. Some misshapen cells were observed to be present in the 10 and 32 mg/L test cultures whilst no intact cells were observed to be visible in the 100 mg/L test cultures.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: After the 72-hour test period all control, 1.0 and 3.2 mg/L test cultures were observed to be green dispersions. The 10 mg/L test cultures were observed to be pale green dispersions whilst the 32 and 100 mg/L test cultures were observed to be clear colorless solutions.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ErC50 (72 h) 1.4 mg/L (95% CI: 1.2 - 1.5 mg/L); EyC50 (72 h) 0.60 mg/L (95% CI: 0.52 - 0.69 mg/L)

Reported statistics and error estimates:

Statistical analysis of the growth rate and yield data was carried out for the control, 1.0, 3.2, 10 and 32 mg/L test groups 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).

Any other information on results incl. tables

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 82% to 102% of nominal and so the results are based on nominal test concentrations only.

Table 1: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.075		1.07E+06
	R2	0.075		1.11E+06
	R3	0.077		1.24E+06
	R4	0.065	-	5.52E+05	-
	R5	0.071		8.11E+05
	R6	0.073		9.42E+05
	Mean	0.073		9.56E+05
	SD	0.004		2.47E+05
1.0	R1	0.078	[7]	1.41E+06
	R2	0.077	[5]	1.32E+06
	R3	0.078	[7]	1.42E+06
	Mean	0.078	[6]	1.38E+06	[44]
	SD	0.001		5.37E+04
3.2	R1	0.075	[3]	1.13E+06
	R2	0.075	[3]	1.09E+06
	R3	0.074	[1]	1.02E+06
	Mean	0.075	[2]	1.08E+06	[13]
	SD	0.001		5.96E+04
10	R1	0.051	30	1.90E+05
	R2	0.050	32	1.82E+05
	R3	0.048	34	1.56E+05
	Mean	0.050	32	1.76E+05	82
	SD	0.002		1.79E+04
32	R1	0.011	85	6.12E+03
	R2	0.008	89	4.07E+03
	R3	0.004	95	1.80E+03
	Mean	0.008	90	4.00E+03	100
	SD	0.004		2.16E+03
100	R1	-0.005	107	-1.46E+03
	R2	-0.005	107	-1.62E+03
	R3	-0.004	105	-1.35E+03
	Mean	-0.005	106	-1.48E+03	100
	SD	0.001		1.37E+02

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1-R6= Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

Table 2: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	192	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	14%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	5%	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.