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Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well described GLP compliant study conducted to recognized international test guidelines
Justification for type of information:
Read-across from supporting substance (structural analogue or surrogate)
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
Concentrations: 0, 4.6, 10, 22, 46, 100, 220 mg/L
Sampling at 0h and 72h
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 220 mg/L was prepared in test medium applying 5-6 minutes of magnetic stirring. Lower test concentrations were prepared by subsequent dilutions of the stock solution in test medium
The test medium source is M2 according to the ISO Standard.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: green algae
- Source: in house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: according to OECD guideline

- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: not indicated
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
24 mg/L as CaCO3
Test temperature:
test medium 8.3 +/- 0.2
during the test (0h and 72h measurements):
at 0 mg/L - 8.1 and 9.3
at 4.6 mg/L - 8.0 and 9.3
at 10 mg/L - 7.7 and 9.2
at 22 mg/L - 7.2 and 9.2
at 46 mg/L - 6.8 and 9.2
at 100 mg/L - 5.0 and 5.2
at 220 mg/L - 3.1 and 3.0
Nominal and measured concentrations:
nominal - 0, 4.6, 10, 22, 46, 100, 220 mg/L
measured - 0, 4.3, 10.1, 21.9, 44.8, 101, 224/233 mg/L
Details on test conditions:
- Test vessel: glass
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass 100mL with 50 mL medium
- Aeration: no
- Initial cells density: 10,000 cells/mL
- Control end cells density: 1,110,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Standard medium used: yes

- Source/preparation of dilution water: M2 according to the ISO Standard
- Culture medium different from test medium: no
- Intervals of water quality measurement: 0h and 72h except temperature which was continuous

- Sterile test conditions: not indicated
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 73-101 uE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : inhibition of cell growth and reduction of growth rate
- Measurement method: not indicated

- Spacing factor for test concentrations: 2X
Reference substance (positive control):
potassium dichromate
72 h
Dose descriptor:
Effect conc.:
70 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
cell number
72 h
Dose descriptor:
Effect conc.:
73 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
ca. 44.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none indicated
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: appx 1 mg/L; with the EC50 a bit higher for growth rate than for biomass inhibition
- Other:

Nominal Conc - 0, 4.6, 10, 22, 46, 100, 220 mg/L

Grow Rate Inhib (% of controls) - 0, -0.3, 1.0, -0.4, -1.3, 84, 98

Biomass Inhib (% of controls) - 0, 0.5, 5.9, 1.3, -4.6, 97, 100

Growth Factor Control - 27.6

The 72h NOEC is 44.8 mg/L active ingredient (a.i.) based on measured concentration

Very possible that the effects observed at the highest test concentrations are related to the acidity of the test substance.

The measured pH values at 0 and 72 hours were:

mg/L        0h       72h

0              8.1       9.3

4.6           8.0       9.3

10            7.7       9.2

22            7.2       9.2

46            6.8       9.2

100          5.0       5.2

220          3.1       3.0

Validity criteria fulfilled:
The EC50 for the test substance (active ingredient) based on growth inhibition (biomass) was 70 mg/L. It is very possible the toxicity was a result of the acidity of the test substance as the pH values were very low at the effect level concentrations. The 72h NOEC is 44.8 mg/L a.i..

Description of key information

NOEC for freshwater algae = 44.8 mg/L.  

Key value for chemical safety assessment

EC50 for freshwater algae:
73 mg/L
EC10 or NOEC for freshwater algae:
44.8 mg/L

Additional information

There is a 2005 good quality "key study" for a 99.8% pure benzenesulfonic acid (CAS No. 98-11-3). It followed OECD Guideline 201 and was done under GLP with analytical confirmation and a reference substance. The 72h-EC50 for Selenastrum capricornutum was 70 mg/L active ingredient based on biomass inhibition. The 72h-EC50 was 73 mg/L for growth rate inhibition. The reported 72h-NOEC was 44.8 mg/L. Both values are based on measured test substance. It appears the pH of the test substance was responsible for the observed toxicity. The report indicates that the observed toxicity may very well have been due to the acidity of the test substance. The pH of the test medium at the two highest test concentrations (100 and 220 mg/L) was in the range of 5 and 3, respectively.The test conditions were confirmed to be valid and the measured concentrations were in the range of 90-110% of the nominal concentrations for the 5 exposures


For read-across purposes, there are three guideline algal toxicity studies for the hydrotropes (i. e., the salt form of the aromatic sulphonic acids) and all three studies indicate toxicity in the same range as for the acid form.

The key study is a 1993 USEPA Guideline study - EPA OTS 797.1500 - Algal Toxicity. The study was not conducted with GLP requirements but considerable detail is available. The freshwater Selenastrum sp. was exposed to a range of nominal concentrations of the test substance, sodium xylene sulphonate (CAS No 1300-72-7). The 96-hour EC50 was 230 mg/L active substance based on cell density (with a 95% CL of 47-1200 mg/L). There was an apparent dose response and the 96-hour NOEC was 31 mg/L. The test substance was observed to be algistatic but not algicidal at the highest concentration, 1000 mg/L active substance.

The 2nd study is a 1994 EPA Guideline study (EPA OTS 797.1500) of calcium xylene sulphonate (CAS No 28088-63-3) conducted under GLP requirements and with analytical confirmation reports a 96h-EC50 of 758 mg/L active substance (with a 95% confidence limit of 592-924 mg/L) and a NOEC of 240 mg/L based on growth rate of Selenastrum sp.

The 3rd study is a 1995 EU Guideline study (EU C.3 Algal Inhibition Test) of sodium toluene sulphonate (CAS No 657-84-1 that is similar to CAS No 12068-03-3) conducted under GLP requirements and with analytical confirmation reports a 72h-EbC50 of 150 mg/L active substance based on cell density and a 72h-ErC50 of 310 mg/L based on specific growth rate of Scenedesmus sp. The 72h-NOEC is 40 mg/L active substance based on biomass.