Z-109

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study; well documented study report; test material measured analytically

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: colorless colourless viscous liquid

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
1) OECD 2000. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures

A determination of the General PhysicoChemical Properties on the test material gave a water solubility value of less than or equal to 0.138 mg/L at 20 degrees C. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation (e.g., ultrasonication and high shear mixing). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions. A saturated solution was prepared from an initial test material dispersion at a concentration of 500 mg/L.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-hour test period all control and test cultures were observed to be pale green dispersions.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: Desmodesmus subspicatus, strain CCAP 276/20
- Source (laboratory, culture collection): Liquid cultures were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation):
- Method of cultivation: Cultures were maintained in the laboratory at a temperature of 21 degrees C under constant aeration and constant illumination.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

This experimental design conforms to a limit test to confirm that at the highest attainable test concentration no effect on growth was observed.

Test conditions

Test temperature:

Temperature was maintained at 24C throughout the test

pH:

The pH values of the control cultures were observed to increase from 7.3 at 0 hours to 7.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the test guidelines. The pH values of the saturated test material concentration were 7.2 at 0 hours and 7.6 and 72 hours.

Salinity:

not applicable.

Nominal and measured concentrations:

Control
Mean Measured 0.45 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
As in the range finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control and 0.45 mg/L treatment group. The control group was maintained under identical conditions but not exposed to the test material. Preculture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.61x105 cells per mL. Inoculation of 2 litres of test medium with this algal suspension gave an initial nominal cell density of 4x103 cells per mL. The flasks were plugged with polyurethane foam bungs and incubated at 24 degrees C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. Samples were taken at 0, 24, 48, and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.


GROWTH MEDIUM
The culture medium used for the range finding and definitive tests was the same as that used to maintain the stock culture. Stock solutions of the culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5.

- Standard medium used: yes

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate as the reference material. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. The results from the positive control with potassium dichromate were wit

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and biomass

Remarks on result:

other: The EC50 values based on the geometric mean measured test concentration were greater than 0.35 mg/L and there were no toxic effects at saturation.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.45 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and biomass

Remarks on result:

other: The EC50 value based on the 0 hour mean measured test concentration gave EC50 values of greater than 0.45 mg/L.

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and biomass

Remarks on result:

other: The No Observed Effect Concentration was 0.35 mg/L the highest test concentration possible and demonstrated that there were no toxic effects at saturation.

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate and biomass

Remarks on result:

other: The No Observed Effect Concentration was 0.45 mg/L and no toxic effects at saturation were observed.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control of test cultures at 72 hours.

Any other information on results incl. tables

Table. Inhibition of Growth Rate and Yield in the Definitive Test

0-Hour Mean Measured Test Concentration (mg/L)	Growth Rate (cells/ml/hour)       0 to 72 hours              % Inhibition		Yield (cells/mL)     0 to 72 hours                % Inhibition
Control R1 R2 R3 R4 R5 R6 Mean SD	0.06 0.056 0.058 0.060 0.061 0.059 0.059 0.002	--	3.03E5 2.17E5 2.51E5 2.96E5 3.1E5 2.72E5 2.75E5 3.57E4	--
0.45 Saturated Solution R1 R2 R3 R4 R5 R6 Mean SD	0.065 0.059 0.064 0.059 0.066 0.070 0.064 0.004	[10] 0 [8] 0 [12] [19] [8]	4.40E5 2.68E5 3.97E5 2.77E5 4.72E5 6.25E5 4.13E5 1.33E5	[50]

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Desmodesmus subspicatus to the test material resulted in no toxic effects at saturation. The EC50 values were greater than the level of saturation, which at 0 hours was measured at 0.45 mg/L and using the geometric mean measured test concentrations over the 72 hour period was greater than 0.35 mg/L. The No Observed Effect Concentration was greater than the level of saturation, which was measured at 0.45 mg/L at time 0 hours.

Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green algaDesmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No. 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

 

Methods:

A determination of the General Physico-Chemical Properties study conducted on the test material gave a water solubility value for the test material of less than or equal to 0.138 mg/L at 20 degrees C. A pre-study media preparation trial indicated that a dissolved test material concentration of approximately 0.1 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

 

Following a preliminary range-finding test,Desmodesmus subspicatuswas exposed to a solution of the test material at a 0 hour mean measured test concentration of 0.45 mg/L for 72 hours, under constant illumination and shaking at a temperature of 24 degrees C. The test material solution was prepared by shaking an excess (500 mg/L) of test material in culture medium in sealed vessels at approximately 300 rpm for 24 hours. Potential undissolved material was removed by centrifugation at 10000 g for 30 minutes. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

 

Results:

Exposure ofDesmodesmus subspicatusto the test material, based on the 0 hour mean measured test concentration gave EC50 values of greater than 0.45 mg/L which is greater than the level of saturation. This study showed that there were no toxic effects at saturation, and the No Observed Effect Concentration was 0.45 mg/L the level of saturation.