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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:

24-27 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Name: NO.148 AQN
Lot No.: 00080
CAS No.: 214115-79-4
Chemical name: 4'-{[3-(diethylamino)propyl]carbamoyl}-9,10-dihydro-9,10-dioxoanthracene-2-carboxanilide
Purity: 96.81 %
Description: Pale yellow powder
Production date: 05 April 2010
Expiry date: 04 April 2012
Storage: room temperature (15 – 25 °C)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Concentrations ofthe test material in the test solution was determined at the beginning and at the end of the study.
At the start of the test five samples was taken from the test solution. At the end of the test four samples were taken from each of the test vessels. Both occasions one sample was taken from the control solution.
- Sample storage conditions before analysis: room temperature (15 – 25 °C)

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:Because the test item is poorly soluble in water, the stock solution used in the test was prepared by a method described below (see Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.):
A stock solution (nominally 100 mg/L) was prepared by mechanical dispersion one day before the start of the test. This solution was shaken for about 24 hours. After shaking the non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100 % v/v saturated solution.
- Controls: yes, algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, GERMANY.
- Age of inoculum (at test initiation): The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 107 cells/mL.
- Method of cultivation: Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of LAB Research Ltd.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

no data

Test temperature:

Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.0 – 22.2 °C measured in the flask and between 21.7 and 22.4 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the study, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.86 – 8.09 at the start and 8.24 – 8.67 at the end of the study.

Dissolved oxygen:

no data

Salinity:

n/a

Nominal and measured concentrations:

Nominal: 1.0, 2.6, 6.4, 16.0, 40.0 and 100.0 % v/v saturated solution.
Measured: 0.01, 0.02, 0.04, 0.10, 0.34 and 0.60 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension.
- Type (delete if not applicable): The flasks were covered with air-permeable stoppers.
- Material, size, headspace, fill volume: 100 mL algal suspension
- Initial cells density: The initial cell number in the test cultures was 10^4 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: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.

Stock solution Substance Final concentration in the prepared growth medium
Stock solution 1 (macro nutrients) NH4Cl 15.0 mg/L
MgCl2 × 6 H2O 12.0 mg/L
CaCl2 × 2 H2O 18.0 mg/L
MgSO4 × 7 H2O 15.0 mg/L
KH2PO4 1.6 mg/L

Stock solution 2 (iron) FeCl3 × 6 H2O 64.0 μg/L
Na2EDTA × 2H2O 100.0 μg/L

Stock solution 3 (trace elements) H3BO3 185.0 μg/L
MnCl2 × 4 H2O 415.0 μg/L
ZnCl2 3.0 μg/L
CoCl2 × 6 H2O 1.5 μg/L
CuCl2 × 2 H2O 0.01 μg/L
Na2MoO4 × 2 H2O 7.0 μg/L

Stock solution 4 (bicarbonate) NaHCO3 50.0 mg/L


OTHER TEST CONDITIONS
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 110 μE/m2/s, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm) and it is checked periodically.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three in the control group.

- Test concentrations: 0, 1, 10, 50 & 100 %v/v saturated solution
- Results used to determine the conditions for the definitive study: Because significant toxic response was observed during the preliminary range-finding test, six test concentrations in a geometric series (factor 2.5) and one control was used in the main test.
The concentrations in the definitive test are based on the results of the preliminary range-finding test and were: 1.0, 2.6, 6.4, 16.0, 40.0 and 100.0 % v/v saturated solution.
The corresponding calculated test item concentrations were: 0.01, 0.02, 0.04, 0.10, 0.34 and 0.60 mg/L.
The test results are based on the calculated test item concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.37-0.51

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.24 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 0.20-0.29

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Thin cells were observed at the highest concentration level of 100 % v/v saturated solution, 48 and 72 hours after the start of the test.

Results with reference substance (positive control):

The date of the last study (Study Code: 10/157-022AL) with the reference item Potassium dichromate is: 22 – 25 June 2010.
The ErC 50 : 1.00 mg/L, (95 % confidence limits: 0.89 – 1.13 mg/L)
The EbC 50 : 0.61 mg/L, (95 % confidence limits: 0.54 – 0.68 mg/L)
The EyC 50 : 0.47 mg/L, (95 % confidence limits: 0.43 – 0.52 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software (based on the calculated geometric mean concentrations).
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Any other information on results incl. tables

VALIDITY

The cell density in the control cultures increased by a factor of 90.33 within three days.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 8.18 %.

The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.05 %.

All validity criteria were met, therefore the study can be considered as valid.

CONCENTRATIONS OF THE TEST ITEM

The concentrations in the definitive test were: 1.0, 2.6, 6.4, 16.0, 40.0 and 100.0 % v/v saturated solution.

The corresponding calculated test item concentrations were: 0.01, 0.02, 0.04, 0.10, 0.34 and 0.60 mg/L.

Since there was greater than 20% difference between the start and end values, the geometric mean was calculated for the two highest concentrations (40.0 and 100.0 % v/v saturated solution). The concentrations were below the Limit of Quantification (LOQ) at the two lowest test groups of 1.0 and 2.6 % v/v saturated solution. Furthermore the concentrations could not quantified at the end of the test at 6.4 and 16.0 % v/v saturated solution. Therefore the concentrations at these cases were extrapolated from the calculated geometric mean value of the highest examined concentration level (0.60 mg/L calculated).

CELL NUMBERS

The cell number in each flask was determined at the 24th, 48th, 72nd hours.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS

Thin cells were observed at the highest concentration level of 100 % v/v saturated solution, 48 and 72 hours after the start of the test.

AVERAGE SPECIFIC GROWTH RATES

The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value in the concentration range of 0.34 – 0.60 mg/L, accordingly the No Observed Effect Concentration (NOEC) was determined as 0.10 mg/L.

The 72 h ErC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.43 mg/L (95 % confidence limits: 0.37 – 0.51 mg/L).

AREAS UNDER THE GROWTH CURVES

The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value in the concentration range of 0.10 – 0.60 mg/L, accordingly the No Observed Effect Concentration (NOEC) was determined as 0.04 mg/L.

The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.24 mg/L (95 % confidence limits: 0.20 – 0.29 mg/L).

YIELD

The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value in the concentration range of 0.10 – 0.60 mg/L, accordingly the No Observed Effect Concentration (NOEC) was determined as 0.04 mg/L.

The 72 h EyC50 value was determined [by Probit analysis (TOXSTAT software)] as 0.26 mg/L (95 % confidence limits: 0.22 – 0.30 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 and EbC50 are 0.43 and 0.24 respectively.
With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the concentration range of 0.34 – 0.60 mg/L; the 0-72 h yield and areas were significantly different from that of the control in the concentration range of 0.10 – 0.60 mg/L. The overall NOEC was determined as 0.04 mg/L; the overall LOEC was determined as 0.10 mg/L.

Executive summary:

The effect of the test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

A significant toxic response was observed during the preliminary range-finding test, therefore six test concentrations in a geometric series (factor 2.5) and one untreated control were tested in the main experiment.

Because the analysed concentrations deviated more than 20 percent from the nominal concentration throughout the test, the biologically results are based on the concentrations calculated from the results of the analytical measurements.

The nominal concentrations of the teat item used in the main experiment were: 1.0, 2.6, 6.4, 16.0, 40.0 and 100.0 % v/v saturated solution. The corresponding calculated test item concentrations were: 0.01, 0.02, 0.04, 0.10, 0.34 and 0.60 mg/L.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates were significantly different from that of the control group in the concentration range of 0.34 – 0.60 mg/L; the 0-72 h yield and areas were significantly different from that of the control in the concentration range of 0.10 – 0.60 mg/L. The overall NOEC was determined as 0.04 mg/L; the overall LOEC was determined as 0.10 mg/L. The ErC50 and EbC50 are 0.43 and 0.24 respectively.