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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:

05-28 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and EC guidelines and according to GLP principles.

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:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Test guideline 3

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

Reference substance name:

No. 338 RED

IUPAC Name:

No. 338 RED

Details on test material:

- Substance type: red powder
- Lot/batch No.: 00004

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations and control
- Sampling method: 3 ml from the approximate centre of the test vessels at t=0, t=24 and t=72 h
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis

Test solutions

Details on test solutions:

All test solutions were individually prepared. Preparation included three days of magnetic stirring in the dark followed by filtration through a 0.45 μm membrane filter (Whatman, RC55) to remove the remaining undissolved material. The final test solutions were all clear and increasingly pink/red with increasing concentration.

After preparation, volumes of 50 ml (combined limit/range-finding test) or 30 ml (final test) were added to each replicate of the respective test concentration. Subsequently, respectively 1.0 or 0.6 ml of an algal suspension was added to each replicate providing a cell density of 10^4 cells/ml.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/ml.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol (24 mg CaCO3/L)

Test temperature:

Between 21 and 23°C

pH:

Between 7.9 and 8.3

Nominal and measured concentrations:

Nominal: Loading rates of 1.0, 3.2, 10, 32 and 100 mg/l (0.45 µm filtered)
Measured: Time Weighted Average (TWA) concentrations corresponded to 0.01, 0.07, 0.18, 0.42 and 0.34 mg/l.

Details on test conditions:

FINAL STUDY:
TEST SYSTEM
- Test type Open, static, with reduced light path and light intensity close to the maximum allowed level to prevent any colour related effect due to light shielding.
- Material, size, headspace, fill volume: 250 ml tissue culture flasks (75 cm2, Greiner), containing 30 ml of test solution.
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 202000 cells/ml
Replicates:
6 replicates of the control and the highest concentration
3 replicates of each lower test concentration
1 or 2 replicates of each concentration without algae

GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using Sylvania TLD-lamps of the type ‘Gro-Lux’ of 30 Watt, with a light intensity within the range of 4929 to 6712 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: control and 1.0, 3.2, 10, 32 and 100 mg/l (0.45 µm filtered)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.098-0.26 mg/l

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.01 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.085 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 0.068-0.11 mg/l

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no

Results with reference substance (positive control):

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.4 mg/l with a 95% confidence interval ranging from 0.97 to 2.0 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.69 mg/l with a 95% confidence interval ranging from 0.48 to 1.0 mg/l. The historical ranges of the 72h-EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 for growth rate was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance reduced growth rate of this fresh water algae species significantly at a TWA concentration of 0.07 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) based on TWA concentrations was 0.16 mg/l with a 95% confidence interval ranging from 0.098 to 0.26 mg/l.

The EC50 for yield inhibition (EYC50: 0-72h) based on TWA concentrations was 0.085 mg/l with a 95% confidence interval ranging from 0.068 to 0.11 mg/l.

The NOEC for growth rate reduction equalled a TWA concentration of 0.01 mg/l. The NOEC for yield inhibition was below 0.01 mg/l.

Note that the present study was performed applying a reduced light path and light intensity close to the maximum allowed level to prevent any colour related effect due to light shielding. Consequently, any inhibition of algal growth can with reasonable certainty be related to toxicity and is not due to light shielding.