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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:

From October 22, 2002 to October 25, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

CAS No.: 078567-28-9; Batch No.: WDJ 1853D-3; Purity: 100 %; Appearance: liquid, viseous, yellowish

Analytical monitoring:

yes

Details on sampling:

Alga cells were checked for any unusual cell shapes, color differences, differences in chloroplast morphology, flocculations, adherence of algae to test containers or aggregation of alga cells.

Vehicle:

no

Details on test solutions:

Dispersions with the nominal loading of 12.5, 25, 50 and 100 mg/L, were prepared with test medium and shaken with 20 rpm tor 24 h (Rotating Shaker 3040, GFL). Finally undissolved particles of the test substance were removed by centrifugation (20 min, 3000 rpm). The dispersion with the nominal loading of 12.5 mg/L served as stock solution for preparation of test loading of 6.25 mg/L. Dilutions were necessary since weighing out of smaller test substance amounts were not possible.
Controls: Six replicates (without test substance) were tested under the same test conditions as the test replicates.

Test organisms (species):

Scenedesmus sp.

Details on test organisms:

Test organism: Scenedesmus subspicatus CHODAT SAG 86.81,
Origin: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen,
Method of cultivation: fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 uE/m2*s for 24 h per day,
Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994).

Test type:

static

Water media type:

other:

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 2 °C

pH:

7.72-8.34

Nominal and measured concentrations:

Nominal concentrations: 6.25 - 12.5- 25 - 50 - 100 mg/L

Details on test conditions:

Replicates: 3 replicates per loading level, 6 per control,
Test container: Plastic cuvettes (0 50 mm), transparent, with top, volume: 20 mL,
Test volume: 10 mL,
Preculture: A four day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with test medium. For the start of the test the preculture was diluted with test medium to receive an initial cell concentration of approximately 1 x 10+E4 cells/mL in the replicates. All algae were from the same source and have not been used in a previous study,
Application: at the test start fluorescence was measured after application of the test substance,
Agitation: test containers were placed on a rotary shaker and oscillated at approximately 70 rpm,
Light intensity: nominally 60 - 120 uE/m2*s,
Light regime: 24 h/d light,
Recovery of algae: after 72 h 0.5 mL alga suspension from the nominal test loading 100 mg/L and control were transferred to 10 mL untreated test medium and allowed to grow for further 3 d to determine whether the effect of the test substance was reversible,
Measurements: Cell density was measured via Chlorophyll-a-fluorescence (excitation at 435 nm, emission at 685 nm). Each replicate was measured 6-fold. The cell density was measured at the beginning of the test and every 24 h. Filtrated culture medium was used as ground signal. The pH-value at the beginning of the test was measured out of two additional replicates of each loading level and control. At the end it was measured from a pool of all replicates. The room temperature was measured continuously by a hygrothermographe. Light intensities were measured prior to test start. Microscopic evaluation of the cells was determined at the start and end of the incubation.

Reference substance (positive control):

yes

Remarks:

Controls: Six replicates (without test substance) were tested under the same test conditions as the test replicates.

Key result

Duration:

72 h

Dose descriptor:

other: EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

EbC5O: 0.49 mg/L (95 % CI: 0.45 - 0.54); ErC50: 0.94 mg/L (95 % CI: 0.82 - 1.07).
The EC50 values of the reference substance potassium dichromate after 72 h met the validity criteria according to EU Method C.3 Annex 2 (preseribed ranges are 0.20 - 0.75 mg/L and 0.60 - 1.03 mg/L potassium diehromate for inhibition of biomass growth and raterelated inhibition, respeetively).

Reported statistics and error estimates:

NOEL values were calculated using One Way Analysis of Variance, DUNNETT'S Method and BONFERRONI t-test (a=0.05).

Validity criteria:

The cell density had to increase at minimum 16-fold in the control replicates within 72 h. The temperature during the test had to be in the range of 21-25 °C controlled at ± 2 °C. The pH-value of the control replicates should not normally deviate by more than 1.0 units during the test.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h EL50 of the test substance in fresh water algae was determined to be greater than 100 mg/L and the 72 h NOELs were determined to be 6.25 mg/L (biomass growth) and 25 mg/L (growth rate).

Executive summary:

A study was conducted to determine the short term toxicity of the test substance to Scenedesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. The study was conducted under static conditions with an initial cell density of 10E+4 cells/mL. 5 loading levels of the water accommodated fraction (WAF) were tested in a geometrical series with a dilution factor of 2: nominal test loading: 6.25 - 12.5 - 25 - 50 - 100 mg/L. Dispersions of the test substance at nominal loading of 12.5, 25, 50 and 100 mg/L were prepared. The dispersion with the nominal loading of 12.5 mg/L served as stock dispersion for preparation of test loading at 6.25 mg/L. Three replicates were tested for each loading level and six replicates for control. Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities. Water quality parameters such as pH, measured at 0 and 72 h, and room temperature, measured continuously, were determined to be within the acceptable limits. The concentrations of DOC were analysed at all loading levels at test start and test end. All effect values are given based on the nominal test loadings. Under the study conditions, the test substance was found to inhibit the growth of the freshwater green alga Scenedesmus subspicatus at nominal loading of ≥12.5 mg/L (biomass growth) and at ≥50 mg/L (growth rate). Under the study conditions, the 72 h EL50 of the test substance in fresh water algae was determined to be greater than 100 mg/L and the 72 h NOELs were determined to be 6.25 mg/L (biomass growth) and 25 mg/L (growth rate) (Scheerbaum, 2002).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

25 mg/L

Additional information

A study was conducted to determine the short term toxicity of the test substance to Scenedesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. The study was conducted under static conditions with an initial cell density of 10E+4 cells/mL. 5 loading levels of the water accommodated fraction (WAF) were tested in a geometrical series with a dilution factor of 2: nominal test loading: 6.25 - 12.5 - 25 - 50 - 100 mg/L. Dispersions of the test substance at nominal loading of 12.5, 25, 50 and 100 mg/L were prepared. The dispersion with the nominal loading of 12.5 mg/L served as stock dispersion for preparation of test loading at 6.25 mg/L. Three replicates were tested for each loading level and six replicates for control. Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities. Water quality parameters such as pH, measured at 0 and 72 h, and room temperature, measured continuously, were determined to be within the acceptable limits. The concentrations of DOC were analysed at all loading levels at test start and test end. All effect values are given based on the nominal test loadings. Under the study conditions, the test substance was found to inhibit the growth of the freshwater green alga Scenedesmus subspicatus at nominal loading of ≥12.5 mg/L (biomass growth) and at ≥50 mg/L (growth rate). Under the study conditions, the 72 h EL50 of the test substance in fresh water algae was determined to be greater than 100 mg/L and the 72 h NOELs were determined to be 6.25 mg/L (biomass growth) and 25 mg/L (growth rate) (Scheerbaum, 2002).