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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:

30 May 2003 to 26 September 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

June 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EC Comission Directive 1999/11/EC of 8 March 1999 (Official Journal No L 77/8)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test concentrations were verified by chemical analysis. Triplicate 100 ml samples were taken from control and test stock solutions at 0 hours and from culture vessels at 72 hours (replicates pooled) for analysis.
Additional samples were taken from flasks containing the "no algae" cultures (see Details on test solutions) at 72 hours (replicates pooled).

Vehicle:

no

Details on test solutions:

The test substance, 200 mg, was dispersed in 2.0 litres of test medium and ultrasonicated for one hour with intermitted vigorous shaking to produce a stock dispersion of 100 mg/l. Serial dilutions of this stock dispersion were prepared with test medium to give the nominal test concentrations (in descending order of concentration) of 22, 10, 4.6, 2.2, 1.0 and 0.46 mg/l.
Additional flasks containing the test substance at a nominal concentration equivalent to 10 mg/l, but without the presence of algal cells, were also prepared in order to obtain information on the extent to which the test substance was lost by either adsorption onto or absorption by the algal cells. These cultures are referred to as the "no algae" test cultures.

Test organisms (species):

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

Details on test organisms:

Name: Pseudokirchneriella subcapitata, Strain No CCAP 278/4
Source: Algal cultures were obtained from the Culture Collection of Algae & Protozoa, CEH, Windermere, Cumbria, UK
Pre-culture: Flasks containing sterile nutrient medium were inoculated from a master culture and were maintained under continuous illumination (7510- 8240 lux) in an orbital incubator at 23.7 to 24.2°C, to give an algal suspension in log phase growth, characterised by a cell density of 2.5 x 10E6 cells/ml
Algal nutrient medium: sterile nutrient medium as recommended in Official Journal No L383A Part C.3 (used for pre-culture and study)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 - 25°C; Mean 24 °C (n=4);
According to the Guideline in force at the time the test was performed, the accepted temperature range was 21 - 25 °C.

pH:

7.8 – 8.7

Nominal and measured concentrations:

Nominal test concentrations: 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/l
Mean measured test concentrations (unfiltered): 0.43, 0.99, 2.0, 4.0, 8.8 and 21 mg/l
Mean measured concentrations (filtered): 0.24, 0.72, 1.8, 3.8, 8.4, 20 mg/l

The unfiltered samples ranged from 86.4 – 97.9 % of nominal (fresh medium; 0 hours) and from 76.8 – 94.0 % of nominal (expired medium; 72 hours). The filtered samples ranged from 76.8 – 94.0 % of nominal (fresh medium; 0 hours) and from 26.6 – 91.1 % of nominal (expired medium; 72 hours).
All results are expressed in terms of mean measured concentrations of unfiltered media. For details on the chemical analysis (measured concentrations of T-71) see Appendix 3 “T-71 – results acute toxicity to algae.pdf”).

Details on test conditions:

Experimental design: Six test concentrations were prepared in triplicate plus one untreated control with six replicates.

Selection of test concentrations: The definitive test concentrations were selected following three range finding experiments and one failed definitive study. The definitive study was initially run using concentrations between 2.2 and 100 mg/l. However, this study failed due to significant growth inhibition at the lowest concentration. Therefore, the definitive study was re-run using concentrations between 0.46 – 22 mg/l with a spacing factor of 2.2.

Culture conditions: Conical flasks (250 ml) each containing 120 ml of test or control culture were loosely stoppered and placed without conscious bias in a Gallenkamp illuminated orbital incubator. The cultures were incubated, without medium renewal, for 72 hours under continuous illumination of 7860 to 8470 lux provided by fluorescent tubes. Gaseous exchange and suspension of the algal cells were ensured by the continuous oscillation of the orbital shaker platform, at 130 revolutions per minute.
Algal nutrient medium: sterile nutrient medium as recommended in Official Journal No L383A Part C.3
Measurement of Growth: Samples were taken at 0, 24, 48 and 72 hours and cell densities were determined by direct counting using a Coulter® Multisizer II particle counter. The pH of the medium in each concentration and controls was measured at the start and the end of the test, the temperature was measured at 0, 24, 48 and 72 hours.

At the end of the test the test and control cultures were inspected microscopically.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: extrapolated value;

Remarks:

95% confidence limits (21.8 - 23.8 mg/l)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits (12 - 16 mg/l)

Details on results:

There was a clear treatment related effect on algal growth, with significant inhibition of biomass and growth rate at concentrations >0.99 mg/l and >2.0 mg/l respectively.
At the highest test concentration (21 mg/l) the inhibition of growth was determined to be 43.3 %. Therefore the EC50 (growth rate) was calculated by extrapolation (see attachment “T-71 – statistical analysis of algal growth inhibition.pdf”).
Comparison of the measured concentrations of the test substance in flasks with and without algae indicated that the presence of algal cells had not significantly affected the stability of the exposure solution at that concentration.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: not significant

For details on results see attached documents "T-71 - results acute toxicity to algae.pdf".

Results with reference substance (positive control):

Reference substance (positive control) not used in this study.

Reported statistics and error estimates:

The area under each growth curve (cell density v time) and the average specific growth rate for each exponentially growing culture were taken to be indices of growth and were calculated following the procedures detailed in Statistical analysis of algal growth inhibition data.
The no-observed effect concentration (NOEC) values for both of the above parameters were determined using Williams' test to compare the percentage inhibition in each treated group with that for the control cultures (see attachment “T-71 – statistical analysis of algal growth inhibition.pdf").
All 95% confidence intervals for EC50 were calculated using the likelihood ratio method (Donaldson and Schnabel, 1985).

Validity criteria fulfilled:

yes

Remarks:

Increase of biomass >16 after 72 hours (mean cell density of controls at 0 and 72 hours: 1.2 x 10E4 and 1.4 x 10E6 cells/ml, respectively)

Description of key information

Effect values for Pseudokirchneriella subcapitata:

NOEC: 0.99 mg/l, E_rC₅₀(0-72 hours): 22.3 mg/l (EU Method C.3, OECD guideline 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

22.3 mg/L

EC10 or NOEC for freshwater algae:

0.99 mg/L