BAP

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

Study period:

4 September 2008 to 22 December 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Description: Dark amber paste

Analytical monitoring:

no

Details on sampling:

Duplicate samples were taken from the control and test solutions (replicates pooled by group) at 0 and 72 hours. The samples were extraced with dichloromethane, filtered through anhydrous sodium sulphate before redissolving in tetrahydrofuran to bring the sample concentrations within the validated calibration range.

Vehicle:

yes

Remarks:

(dimethylformamide)

Details on test solutions:

Information provided by the Sponsor indicated that the test material was insoluble in cold water. Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 2.0 mg/L (by visual inspection) was obtained using a preliminary solution in dimethylformamide. Based on this information the test material was categorised as being a 'difficult substance' as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

Given the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/I) of test material in culture medium for a period of 24 hours prior to removing any undissolved test material present by filtration (0.2 μm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test material.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 24 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± I °C until the algal cell density was approximately I 0(4) - 10(5) cells/ml.

Test type:

static

Water media type:

other: OECD Culture Medium

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1°C throughout the test.

pH:

The pH values of the control cultures were observed to increase from pH 7.4- 7.5 at 0 hours to pH 7.6 - 7.7 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.

At the start of the test the pH of the test cultures was observed to decrease with increasing test concentration (pH 7.3 at 0.13 mg/1 to pH 5.1 at 3.1 mg/1). This decrease in pH was considered to be due to an intrinsic property of the test material and as such no attempt was made to alter the pH prior to the start of the test.

Nominal and measured concentrations:

0-hour measured concentrations: 0 (control), 0.13, 0.26, 0.58, 1.2 and 3.1 mg/L
Geometric mean measured concentrations: 0 (control), 0.044, 0.10, 0.29, 0.64 and 1.8 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The effect of the test material on the growth of Desmodesmus subspicatus was investigated over a 72-Hour period and, based on the 0-hour measured test concentrations, gave an ErC50 (0 - 72 h) of 1.1 mg/L (95% confidence limits, 1.0 - 1.3 mg/L), a 72-hour EyC50 of 0.64 mg/L (95% confidence limits, 0.57 - 0.73 mg/L), and an EbC50 (0 - 72 h) of 0.64 mg/L (95% confidence limits, 0.58 - 0.72 mg/L). The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 1.2, 1.2 and 0.58 mg/L, respectively, and the No Observed Effect Concentrations were 0.58, 0.58 and 0.26 mg/L respectively.

Based on the geometric mean measured test concentrations, the ErC50 (0 - 72 h) value was 0.59 mg/L (95% confidence limits, 0.52 - 0.68 mg/L), the 72-hour EyC50 value was 0.32 mg/L (95% confidence limits, 0.28 - 0.37 mg/L) and the 72-hour EbC50 value was 0.32 mg/L (95% confidence limits, 0.29 - 0.37 mg/L). The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.64, 0.64 and 0.29 mg/L respectively, and the No Observed Effect Concentrations were 0.29, 0.29 and 0.10 mg/L respectively.

The significant decline in measured test concentrations was observed over the test period was attributed to adsorption to algal cells, as the test material was show to be stable in aqueous medium. It was concluded that the algal cells were exposed to higher concentrations of the test material throughout the exposure period and the key results are therefore based on the 0-hour measured concentrations.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate (ErC50 = 0.57 mg/L; EyC50 = 0.32 mg/L; and EbC50 = 0.31 mg/L) were within the normal range for this reference material.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Validity criteria fulfilled:

yes

Conclusions:

Based on 0-hour measured concentrations, the 72-hour effects on growth rate (ErC50), yield (EyC50) and biomass integral (EbC50) were determined to be 1.1 mg/L, 0.64 mg/L and 0.64 mg/L, respectively. The Lowest Observed Effect Concentration (LOEC) and No Observed Effect Concentration (NOEC) values for specific growth rate after 72 hours were 1.2 mg/L and 0.58 mg/L, respectively.

Executive summary:

A study was performed in accordance with the standardized guideline OECD 201 under GLP conditions to assess the effect of the test material on the growth of the green alga, Desmodesmus subspicatus.

 

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to solutions of the test material at 0-hour measured concentrations of 0.13, 0.26, 0.58, 1.2 and 3.1 mg/L (three replicate flasks per concentration) and a concurrent control (six replicates) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

 

The test material solutions were prepared by stirring an excess of test material in OECD culture medium at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration to produce a saturated solution of the test material with a measured concentration of 3.1 mg/L. This saturated solution was then further diluted as necessary, to provide the remaining test solutions.

 

A positive control was conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material (three replicate flasks per concentration) with a concurrent control (six replicates) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

A decline in measured test concentrations was observed at 72 hours in the range of 0.015 to 1.1 mg/L. A significant decline in measured test concentrations was observed over the test period was attributed to adsorption to algal cells, as the test material was show to be stable in aqueous medium.It was concluded that the algal cells were exposed to higher concentrations of the test material throughout the exposure period and the key results are therefore based on the 0-hour measured concentrations.

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an ErC50 (0 - 72 h) value of 1.1 mg/L (95% confidence limits, 1.0 - 1.3 mg/L). The Lowest Observed Effect Concentration based on inhibition of growth rate was 1.2 mg/L and the No Observed Effect Concentration was 0.58 mg/L.

 

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an EyC50 (0 - 72 h) value of 0.64 mg/L (95% confidence limits, 0.57 - 0.73 mg/L). The Lowest Observed Effect Concentration based on yield was 1.2 mg/L and the No Observed Effect Concentration was 0.58 mg/L.

 

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an EbC50 (0 - 72 h) value of 0.64 mg/L (95% confidence limits, 0.58 - 0.72 mg/L). The Lowest Observed Effect Concentration based on inhibition of biomass integral was 0.58 mg/L and the No Observed Effect Concentration was 0.26 mg/L.

 

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.13 to 3.1 mg/L. The results of the chemical analysis at 0 hours showed measured concentrations to be less than the value obtained during the pre-study media preparation trial. This was considered to be due to the test material forming an emulsion which may have passed through the filter during the media preparation trial which would have resulted in significantly higher dissolved test material concentrations. Given that a toxic response was observed to the measured test concentrations obtained this was considered to have had no adverse effect on the outcome of the test.

 

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an ErC50 (0 - 72 h) of 0.57 mg/L (95% confidence limits 0.48 - 0.66 mg/L), an EyC50 (0 - 72 h) of 0.32 mg/L (95% confidence limits, 0.29 - 0.35 mg/L), and an EbC50 (0 - 72 h) of 0.31 mg/L (95% confidence limits 0.28 - 0.35 mg/L). The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/L respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/L respectively.

Description of key information

In an OECD Guideline 201 study, conducted according to GLP, the effect of the test substance on the growth of the alga, Desmodesmus subspicatus, was investigated and, on 0-hour measured concentrations, the 72-hour effects on growth rate (ErC50), yield (EyC50) and biomass integral (EbC50) were determined to be 1.1 mg/L, 0.64 mg/L and 0.64 mg/L, respectively. The Lowest Observed Effect Concentration (LOEC) and No Observed Effect Concentration (NOEC) values for specific growth rate after 72 hours were 1.2 mg/L and 0.58 mg/L, respectively (Harlan Laboratories, Ltd, 2008).