1,1'-{(phenylmethanediyl)bis[(3-substituted benzene-4,1-diyl)diazene-2,1-diyl{1-[3-(dimethylamino)propyl]-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridine-5,3-diyl}]}dipyridinium chloride lactate salts

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:

February 09-April 18, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study compliant with TG guideline and conducted under GLP in recognized contract research organization. Existing analytical deficiencies do not impair the overall conclusion from the data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test samples for analysis were taken from all test concentrations and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=96 h
Volume 2 ml
Storage Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of test solutions started with a stock solution of 8.6 mg/l (10.1 mg test substance was weighed in a volume of 1 litre; a correction factor of 1.16 was used to correct for water and minor impurities). The lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium. The final test solutions were all clear and increasingly orange with increasing concentration.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: continuous shaking

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

24-hour dark period to ensure ‘worst-case’ exposure to original test substance in case of light sensitive substances, followed by a 72-hour light test period.

Post exposure observation period:

Microscopic observations: in a pre-test withthe test substance algae were observed to be normal (similar to control) after exposure to the test substance.

Test conditions

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

The temperature of the test medium was 22.7°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.2 and 23.7°C.

pH:

7.6-8.3

Dissolved oxygen:

not applicable

Nominal and measured concentrations:

nominal concentrations: 0 (controls), 0.022, 0.067, 0.20, 0.60, 1.8 and 5.4 mg/L
measured concentrations: see Table 1

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml tissue culture flasks (75 cm2, Greiner), containing 30 ml of test solution
- Initial cells density: 10'000 cells/ml
- Control end cells density: 2'993'000 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: synthetic M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continously
- Light intensity and quality: 103 to 120 µE.m-2.s-1; Sylvania TLD-lamps of the type ‘Gro-Lux’ of 30 Watt

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell density (at 0, 24, 48, 72 and 96 hours exposure)
- Determination of cell concentrations: counting chamber (0 h), spectrophotometer (24-96 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.0
- Range finding study
- Test concentrations: 0, 0.1, 1.0, 10, 100 mg/l
- Results used to determine the conditions for the definitive study: see Table 2

Reference substance (positive control):

yes

Remarks:

potassium dichromate, 0, 0.18, 0.32, 0.56, 1.0, 1.8, 3.2 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 0.11-0.30 mg/l

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.031 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

biomass

Remarks on result:

other: 95% CL 0.025-0.039 mg/l

Effect concentrations 3

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

other: growth rate and biomass

Effect concentrations 4

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

0.028 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 0.018-0.046 mg/l

Effect concentrations 5

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

biomass

Remarks on result:

other: 95% CL 0.018-0.046

Effect concentrations 6

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

0.045 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 0.028-0.073

Effect concentrations 7

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

0.024 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

biomass

Remarks on result:

other: 95% CL 0.019-0.029

Details on results:

- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ERC50 (0-72h) 1.84 mg/l with a 95 % confidence interval ranging from 1.37 to 2.46 mg/l

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of the EC50 ,EC10 and EC20 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance. Regression analysis was performed applying concentrations showing a reasonable linear interaction.
Regression line for EC-values for growth reduction: Y=50.24X+87.63 (multiple R=0.9835, n=15)
Regression line for EC-values for yield inhibition: Y=250.34X+427.14 (multiple R=0.9510, n=12)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Criteria of OECD Guideline 201: Increase of biomass >16 (within 3 days after a lag phase of 1 day (dark period)); mean coefficient of variation. for section-by-section specific growth rates ≤35%; coeff. of var. for average specific growth rates ≤7%.

Conclusions:

For classification purposes: 0.1 mg/l < 72h-EC50 ≤ 1 mg/l (based on growth)