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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-09-26, 1995-11-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1981
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Version / remarks:
1989
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 23: Aquatic Toxicity Testing of Difficult Substances and Mixtures
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 200 and 100 mg/L (nominal)
- Sampling method: Filtration of test medium samples (10 mL) taken at the start of the tests, 24, and 72 hours of exposure
- Sample storage conditions before analysis: Frozen (-80 °C)
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection):
- Age of inoculum (at test initiation): Exponentially growing algal suspensions
- Method of cultivation: ISO-medium
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
22.5 - 24 °C
pH:
8.0 - 8.4
Nominal and measured concentrations:
Water-accomodated fractions were prepared with a loading rate of 100 and 200 mg/L. Measured concentrations were at most 2 mg/L. The test substance is rapidly degarded by photolysis.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: Glass, 100mL
- Aeration:
- Initial cells density: 1 x 10E4 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium, except for the FeCl3-6(H2O) content: 80 µg/L instead of 64 µg/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q, tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 6500 - 8000 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Counting chamber (at the begining of the study); spectrophotometer at 720 nm (thereafter)

TEST CONCENTRATIONS
- Rough filtrates (ca. 5 µm) of supersaturated solutions of 100 and 200 mg/L stirred for ca. 4 days.
- Justification for using less concentrations than requested by guideline: The substance is insoluble in water (< 0.006 g/L at 20 °C).
- Range finding study: A measured concentration of 4.7 mg/L was found in the sample taken from the filtrate. Instead of testing dilutions containing 0.1 to 100 % of the filtrate, test solutions contained 0.05 to 50 % of the filtrate yielding a test substance concentration range from 0.002 to 2.35 mg/L. No inhibition of algal growth was observed at any of the test concentrations.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No acute toxic effects within the range of solubilty.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: No acute toxic effects within the range of solubilty.
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Any stimulation of growth found in any treatment: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: see tables 1 and 2
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 0.98 mg/L (growth inhibition) 1.1 mg/L (growth rate reduction)

Table 1. Concentrations of the test substance in test medium (final study I).

Time of sampling (h) Concentration
Code Nominal (mg/L) Analysed1 (mg/L)
0 Sol.l 100 2.02
1002 1.88
Sol.2 200 1.52
2002 1.50
24 Sol.l 100 0.15
1002 0.36
Sol.2 200 0.20
2002 0.13
72 Sol.l 100 0.23
1002 0.36
Sol.2 200 0.18
2002 0.21

1. Concentration analysed in filtrate

2. Samples without algae

Table 2. Concentrations of the test substance in test medium (final study II).

Time of sampling (h) Concentration
Code Nominal (mg/L) Analysed1 (mg/L)
0 Sol.l 100 0.22
1002 0.18
Sol.2 200 0.98
2002 0.83
24 Sol.l 100 0.07
1002 0.10
Sol.2 200 0.23
2002 0.47
72 Sol.l 100 0.07
1002 0.08
Sol.2 200 0.11
2002 0.11

1. Concentration analysed in filtrate

2. Samples without algae

Table 3. Mean cell densities1

Initial concentration (mg/L) Mean cell densities during exposure (x104 cells/mL)
0 h 24 h 48 h 72 h
RF 0.0 1.00 3.57 16.0 61.3
2.35 1.00 5.00 19.4 63.7
Test I 0.0 1.00 4.82 22.5 59.7
2.0 1.00 5.35 17.0 30.3
1.5 1.00 5.57 13.5 24.7
Test II 0.0 1.00 4.21 15.9 50.2
0.2 1.00 4.85 19.4 52.3
1.0 1.00 5.21 18.9 51.6

1. Values are rounded off by the program used for calculations

Table 4. Percentage inhibition of cell growth and percentage reduction of growth rate.

Initial concentration (mg/L) Cell growth(0-72h): Growth rate(cells/mL/h1):
Area (A) mean Inhibition (%) 0-72h mean (μ1) Reduction (%)
RF 0.0 1146.46
0.05714
2.35 1289.88 -12.5 0.05769 -1.0
Test I 0.0 1312.50
0.05649
2.0 840.83 35.9 0.04618 18.3
1.5 693.14 47.2 0.04429 21.6
Test II 0.0 1025.23
0.05439
0.2 1150.73 -12.2 0.05495 -1.0
1.0 1137.07 -10.9 0.05475 -0.7

1. µ = (ln Nn - ln N1)/(tn -t1)

Validity criteria fulfilled:
yes
Conclusions:
No acute toxic effects within the range of solubilty. The 72 hour EC50 (growth rate) of the test material to Pseudokirchneriella subcapitata was determined to be >2 mg/l (above the limit of solubility).
Executive summary:

In this guideline (OECD 201) study conducted with GLP certification, the 72 hour EC50 of the test material (EC 404-360-3) to algae was determined to be >2 mg/l. The study was conducted as a static, limit test (freshwater), using a Water Accomodated Fraction (WAF), on Pseudokirchneriella subcapitata. No acute toxicological effects were observed over the duration of the test. The result of the test does not trigger classification of the test material as hazardous to the aquatic environment under the criteria of the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Description of key information

 Study carried out according to guideline and with GLP compliance. 

Key value for chemical safety assessment

Additional information

One reliable GLP guideline study according to OECD 201 is available (NOTOX 1996). Due to the low solubility of the test item saturated solutions with 100 and 200 mg/L test substance nominal were used. No inhibition of algal growth was observed during a range-finding test at initial exposure concentrations ranging from 0.002 to 2.35 mg/l. In a subsequently performed limit test, significant inhibition of algal growth was recorded in the treated solutions (18-22 %). The initial test concentrations were 1.5 and 2.0 mg/l. There was a relatively high variation between the extinction values of the different replicates, including those recorded in the control replicates. Therefore it was decided to repeat the test.

Concentrations measured at the start of a second limit test ranged from 0.18 to 1.0 mg/l. This time there was little variation between the extinction values of the untreated replicates and no inhibition of cell growth was recorded in any of the treated solutions.

Hence, based on the results of the present studies with Selenastrum capricornutum the test substance did not inhibit cell growth in saturated solutions with nominal concentrations far above the maximum solubility in water.

The EC50 values for both cell growth inhibition (EbC50: 0-72 h) and growth rate reduction (ErC50: 0-72 h) were greater than the maximum attainable concentration of ca.2 mg/l.