Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

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:

from 2008-08-15 to 2008-08-18

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

not applicable

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the start of the study two samples were taken from the control medium and from 0.008-0.016-0.031-0.063 μg/ml concentration level; three samples were taken from 0.125—0.25 μg/ml concentration level. At the end of the study two samples were taken from all concentration levels, as well as from the control medium. The samples were analysed directly after sampling using HPLC-UV method.
The samples were diluted twofold or concentrated depending on the concentration of the test solution (50 ml amount of the test solution was evaporated after freezing on Vacuum concentrator and the test item was redissolved in 2 ml dilution mixture).

Test solutions

Vehicle:

no

Details on test solutions:

Preparation according to OECD guideline 201 (adopted 23rd March 2006)

Test organisms

Test organisms (species):

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

Details on test organisms:

Test System
Species: Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) (Printz-Starr).
Origin: The algae were supplied by the Georg-August-Universität Göttingen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften Experimentelle Phykologie und Sammlung von Algenkulturen (SAG), Nikolausberger Weg 18, D-37073 Göttingen, Germany
Breeding Conditions:
The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days (the pre-culture was incubated for three days).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Test conditions

Hardness:

no data

Test temperature:

23.0 – 23.3 °C

pH:

pH 8.83 to 9.04 at test start
pH 8.78 to 9.83 at test end

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Test Item Concentrations
Based on the results of the preliminary tests six different concentrations arranged in a geometric series and an untreated control group was tested in the main study. The nominal test item concentrations were 0.008; 0.016; 0.031; 0.063; 0.125 and 0.25 mg test item/L. The measured test item concentrations were out the ±20 % range of the nominal concentrations at the start (65 % - 91 %) and at the end (5 % - 27 %) of the test. Therefore, all reported biological results are related to the geometric mean of the measured test item concentrations at the start and at the end of the test calculated by EXCEL Software Program.
The corresponding measured mean test item concentrations were: 0.004; 0.006; 0.012; 0.017; 0.024 and 0.052 mg/L

Details on test conditions:

Dosage of Test Item
In this aquatic toxicity test the stock solution was prepared according to the WAF method (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000). The achieved WAF concentration can comprise the dissolved test material plus any un-dissolved fine test material particles that can cross a paper filter. Hence the achieved total concentration by this method can be significantly higher than the limit of water solubility, which does not include any fine particulates. In the method for water solubility study a fine filter (0.22 μm) was used. In addition, the specific composition of the test medium used (OECD Medium) may possibly result in a different solubility to that in distilled water. A supersaturated test item stock solution (nominally 1000 mg/L) was prepared by dispersing/dissolving the test item amount without the use of any organic solvent into the Test Medium (OECD Medium) two days before the start of the study (on day -2). This solution was shaken for 24 hours at 30 ºC, then was left settling for 24 hours at 20 ºC and thereafter filtrated through a paper filter. The test solutions were prepared by the appropriate diluting of the stock solution on day 0.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits: 0.039 – 0.054

Effect concentrations 2

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

0.143 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits: 0.077 – 0.264

Effect concentrations 3

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % conf. limits: 0.039 – 0.055

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 9

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The nominal test item concentrations were 0.008; 0.016; 0.031; 0.063; 0.125 and 0.25 mg test item/L. The measured test item concentrations were out the ±20 % range of the nominal concentrations at the start (65 % - 91 %) and at the end (5 % - 27 %) of the test. Therefore, all reported biological results are related to the geometric mean of the measured test item concentrations at the start and at the end of the test calculated by EXCEL Software Program. The corresponding measured mean test item concentrations were: 0.004; 0.006; 0.012; 0.017; 0.024 and 0.052 mg/L
Biological Results
Growth Inhibition:
The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.0168 – 0.0522 mg test item/L (based on the measured mean concentrations). The test concentration of 0.0168 mg/L (measured mean) was determined as the 72-hour LOEC. The test concentration of 0.0124 mg/L (measured mean) was determined as the 72-hour NOEC.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 0 h, 24 h, 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399). Percentage inhibition of area A, growth rate r and yield Y were calculated using EXCEL for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399). The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the statistical software program “TOXSTAT 3.5”. For the determination of the LOEC and NOEC, the calculated mean biomass b (area under the growth curve), growth rates r and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test and Williams’test by TOXSTAT software.

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with F 213 Red, the 72-h EC50 based on growth rate was determined as 0.143 mg/L. The 72-h EC50 based on yield was 0.046 mg/L. The overall NOEC was determined to be 0.012 mg/L (all based on the measured mean concentrations).

Executive summary:

The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. The test method of application and the test species Pseudokirchneriella subcapitata are recommended by the test guidelines.

Nominal test item concentrations: 0.008; 0.016; 0.031; 0.063; 0.125 and 0.25 mg/L

Measured test item concentrations (mean): 0.004; 0.006; 0.012; 0.017; 0.024 and 0.052 mg/L

Analytical results

The test item concentration of the test solutions was measured by HPLC-UV method at the start and at the end of the test.

The analytically determined test item concentrations in the analysed test media varied in the range from 65% - 91% at the start and from 5% - 27% at the end of the study in comparison to the nominal value (see the attached analytical report). The deviation of the measured concentrations from the nominal values was higher than 20 %. Therefore, all reported biological results are related to the geometric mean of the measured test item concentrations at the start and at the end of the test calculated by EXCEL Software Program.

Biological Results

F 213 Red had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.0168 – 0.0522 mg test item/L (based on the measured mean concentrations). The test concentration of 0.0168 mg/L (measured mean) was determined as the 72-hour LOEC. The test concentration of 0.0124 mg/L (measured mean) was determined as the 72-hour NOEC.

The 72-h EC50 based on growth rate was determined as 0.143 mg/L, the 72-h EC50 based on yield 0.046 mg/L. The overall NOEC was determined to be 0.012 mg/L (all based on the measured mean concentrations).