Pyridinium toluene-4-sulphonate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Dec 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Mar 2006 (corrected Jul 2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). For the 72-hour stability samples, the contents of the respective replicates were combined prior to sampling.
All samples were stored frozen (at about -20 ± 5 °C) immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An aqueous solution with a nominal content of 80 % was prepared prior to the test. The test substance content of this working solution was analytically verified by HPLC to be 77.9 % (water-free).
A stock solution of the nominal concentration of 52 mg/L was prepared by completely diluting 67 mg of the working solution (corresponding to 52.2 mg water-free test substance) in 1000 mL of test water by intensive stirring at room temperature for 30 minutes in the dark.
The stock solution was serially diluted with test water to prepare the test media of the two highest test concentrations. The test media of the lower test item concentrations were prepared in a series of further dilution steps.
The test media were prepared just before the start of the test.
- Controls: test water

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Source (laboratory, culture collection): SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).

- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
- Method of cultivation: standardized conditions according to the test guidelines


ACCLIMATION
- Acclimation period: four days (pre-culturing)
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.15 mM (15 mg CaCO3/L)

Test temperature:

23 °C

pH:

test start (0 h): 7.5
test end (72 h): 7.5 - 7.7

Nominal and measured concentrations:

nominal concentrations: 0.241, 0.522, 1.10, 2.40, and 5.20 mg/L
measured concentrations:
start: 100 - 108 % of nominal
end (72 h): 103 - 109% of nominal
The biological results are based on the nominal concentration of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 75 mL Erlenmeyer flasks containing 30 mL test medium, covered with a glass lid
- Aeration: continuous shaking on an orbital shaker
Initial cells density: 5000 cells/mL (corresponding to 0.46 x 10E4 relative fluorescence units, RFU)
- Control end cells density: 129 x 10E4 RFU

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The light intensity was measured at the start of the test. The pH was measured and recorded in each treatment at the start and end of the test. The temperature in the incubator was monitored and recorded continuously. The appearance of the test media was also visually controlled and recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously illumination by LED light installed above the test flasks.
- Light intensity and quality: The light intensity at the level of the test solutions was approximately 75 µE s-1 m-2 (range: 74 to 76 µE s-1 m-2, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : daily determination of the algal biomass


- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]

fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK) at an excitation of 440 nm and emission of 680 nm. The measurements were performed at least in duplicate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
The test concentrations in this study were based on the results reported in endpoint study record "Toxicity to aquatic algae and cyanobacteria, supporting study") to refine the results obtained

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corrected for water-content

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence interval: 2.7 - 3.3 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corrected for water-content

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence interval: 0.28 - 0.40 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

corrected for water-content

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: no significant stimulation of growth

All test media were clear solutions throughout the test period.

For details see attached document "Tables Cell densities and inhibition of growth (key study).pdf"

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-hour ErC50: 1.1 mg/L (within the range 0.9 – 1.5 mg/L as recommended by the guidelines)

Reported statistics and error estimates:

Statistical analysis was performed using ToxRat Professional® (ToxRat® Solutions GmbH, Version 3.2.1).
The 72-hour ECx values and their 95 % confidence intervals were calculated by Probit analysis using linear maximum likelihood regression
For the determination of the LOEC and NOEC, Williams’ t test or Welch’s t-test, where appropriate, were used.
All treatments included in the statistical calculations.

Validity criteria fulfilled:

yes

Remarks:

Increase of biomass >16 after 72 hours; mean coefficient of variation for section-by-section specific growth rate <35%; coefficient of variation for average specific growth rate <7%.

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h, (OECD 201, EU C.3):

EC50: 3.0 mg/L, EC10: 0.34 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

3 mg/L

EC10 or NOEC for freshwater algae:

0.34 mg/L