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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-Nov-2009 to 21-Nov-2009
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed carefully and according to internationally accepted test guidelines. However, the test substance could not be detected in any sample and the correlation between fluorescence and biomass was not fully established.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
according to guideline
other: EU Commission Directive 92/69/EEC, C.3: Algal Inhibition Test Commission Regulation (EC) No 440/2008, C.3: Algal Inhibition Test. Also, design and execution of the study were based in part on OECD document 23, Guidance Document on Aquatic Toxicity Tes
GLP compliance:
yes (incl. QA statement)

Test material

Details on test material:
- Name of test material (as cited in study report): MTDID 8191
- Substance type: UVCB material
- Physical state: Yellowish viscous liquid
- Concentration of oligomers: ≥99.0%
- Purity test date: 21 January 2010
- Lot/batch no.: MK 12/048
- Expiration date of the lot/batch: 27-Jan-2010
- Stability under test conditions: Not reported
- Storage condition of test material: In the refrigerator at about 4 °C
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analog

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: After manual shaking, withdrawal of aliquot before inoculation and at end of test (72-hours). Samples diluted with an equal volume of acetonitrile.
- Sample storage conditions before analysis: Samples analyzed immediately

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dispersions were stirred for 96 hours at room temperature in the dark to dissolve a maximum amount of the test item in the dispersion. After stirring, the dispersions were left to settle for 1 hour and the clear phase of each dispersion was then filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm). The negative pressure of the filtration unit was reduced as much as possible to avoid losses of volatile components of the test substance during filtration. The undiluted filtrates were tested as water accommodated fractions (WAFs).
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany
- Age of inoculum (at test initiation): Four days, diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase for inoculation
- Method of cultivation: Algae cultivated under test conditions
- Acclimation period: None

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

24 mg/L as CaCO3
Test temperature:
22 °C
Nominal and measured concentrations:
Nominal concentrations as water-accommodated fractions: 3.2 mg/L, 10 mg/L, 32 mg/L, 100 mg/L.
Concentrations of analyte were below limit of detection in all test vessels.
Details on test conditions:
- Test vessel: 50 mL Erlenmeyer flasks
- Type (delete if not applicable): covered with a glass dish
- Material, size, headspace, fill volume: 50 mL flasks containing 15 ml test medium
- Aeration: Continuous stirring by magnetic stirrers
- Initial cells density: 10,000 cells/mL
- Control end cells density: Not determined
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates):six
- Standard medium used: yes, OECD TG 201 medium.
-Water quality not reported
- Sterile test conditions: yes
- Adjustment of pH: none
- Photoperiod: continuous light
- Light intensity and quality: 8300 Lux (range: 7550 to 8840 Lux, within ±15% of average intensity). Daily randomized repositioning of test flasks

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Fluorimetric measurements (at least duplicate measures) at 0, 24, 48, 72 hours
- Instrumentation: BIO-TEK® Multi-Detection Microplate Reader, Model FLx800. The system is validated and calibrated once per year by system specific tests (“Corners-Test” and “Sensitivity-Test”). Details of validation and calibration not provided.
- Sampling: Small volume taken daily without replacement.
- Chlorophyll measurement: not directly analyzed or reported
- Other: Relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells were assessed by microscopy at end of test for highest exposure level only.
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: flat dose response in preliminary study.
- Range finding study: yes, non-GLP, details not reported
Reference substance (positive control):

Results and discussion

Effect concentrations
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
other: water accommodated fraction
Basis for effect:
growth rate
Remarks on result:
other: Effect measured by fluorimetry
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): Yes, no abnormalities found
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: tested water accommodated fraction. 1 mg/L loading rate was made by dilution of 3.2 mg/L loading rate, rather than as an individual WAF, because of imprecise weight measurement at the 1 mg/L loading rate.
Reported statistics and error estimates:
The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 10E+03) (Table 2). Growth rate and yield were calculated for each test flask. From these, the mean values for growth rate (Table 3) and yield were calculated for each treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data.

The 72-hour EL50 values for the inhibition of average growth rate and yield was established by direct inspection of the data (Tables 2, 3).

For the determination of the Lowest Observed Effect loading Rate (LOELR) and No Observed Effect loading Rate (NOELR), the growth rate and yield at the test concentrations were compared to the control values by Dunnett’s tests. The 72-hour LOELR was 3.2 mg/L. The reported 72-hour NOELR based on both growth rate and yield was 1.0 mg/L.

In the control the biomass increased by a factor of 163 over 72 hours. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was reported as 26%. Additional evaluation by the sponsor found that a mean of section-specific CoVs was 3.7%.

The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 2.1%. Therefore, validity criteria from the OECD test guideline (increase by factor of 16, <35% section by section variance, <7% average specific growth rate variance) were fulfilled.

Any other information on results incl. tables

The test substance was below limit of quantification (<1.01 mg/L) in all test vessels. Therefore, toxicity was assessed based on loading rates of the WAFs tested.


The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

Table 2, Biomass of algae during toxicity testing of the substance withPseudokirchneriella subcapitata
Treatment / Loading rate (mg/L) Rep. no. Biomass of algae1
24 hours 48 hours 72 hours
Control 1 8.6 70.1 447.9
2 8.5 74.0 410.6
3 8.4 75.2 413.8
4 8.5 75.5 367.9
5 8.1 70.5 331.1
6 9.0 75.6 372.6
Mean 8.5 73.5 390.6
SD 0.3 2.5 41.5
1.0 1 8.0 59.4 313.6
2 8.1 70.2 417.7
3 8.4 75.9 331.3
Mean 8.2 68.5 354.2
SD 0.2 8.4 55.7
3.2 1 9.1 63.2 162.2
2 8.9 61.9 179.8
3 10.4 46.3 143.8
Mean 9.5 57.1 161.9
SD 0.8 9.4 18.0
10 1 8.3 43.6 113.8
2 7.3 42.2 122.8
3 7.3 48.4 120.7
Mean 7.6 44.7 119.1
SD 0.6 3.3 4.7
32 1 9.1 65.7 176.5
2 8.0 66.8 191.2
3 9.1 60.8 175.6
Mean 8.7 64.4 181.1
SD 0.6 3.2 8.8
100 1 7.6 60.5 168.4
2 8.3 62.5 179.4
3 7.3 57.6 164.5
Mean 7.7 60.2 170.8
SD 0.5 2.5 7.7
1, The biomass was determined by fluorescence measurement and is given as relative fluorescence units (x 103). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 2.39 x 103relative fluorescence units.

Table 3, Growth rates (day-1) and inhibition of the growth rates (Ir) during toxicity testing of the test substance withPseudokirchneriella subcapitata
Treatment / Loading rate (mg/L)  Growth rates (day-1) and inhibition of the growth rates (Ir)
Section-by-section Average
0-24 h 24-48 h 48-72 h 0-72 h
µ Ir(%) µ Ir(%) µ Ir(%) µ Ir(%)
Control 1.27 0.0 2.15 0.0 1.67 0.0 1.70 0.0
1.0 1.23 3.2 2.12 1.6 1.64 1.6 1.66 2.0
3.2 1.37 -8.2 1.79 16.9 1.05 37.1 1.40* 17.3
10 1.16* 8.7 1.77 18.0 0.98 41.2 1.30* 23.2
32 1.29 -1.7 2.00 7.1 1.03 38.0 1.44* 15.0
100 1.17 7.5 2.05 4.8 1.04 37.5 1.42* 16.2
*, value significantly lower than in the control (according to Dunnett’s-tests, one-sided, α = 0.05). Not tested for sections 24-48 h and 48-72 h.

Applicant's summary and conclusion

Validity criteria fulfilled:
The test substance could not be quantified in solution (LOQ = 1.01 mg/L). The EL50 based on nominal concentrations of water accommodated fractions was >100 mg/L.
Executive summary:

The test substance (CAS# 1101874-33-2) is a UVCB substance of low water solubility. Therefore, toxicity of the substance to algae was determined using water-accommodated fractions (WAFs). The test substance showed similar, statistically-significant growth inhibition in all WAFs ≥ 3.2 mg/L, but 50% inhibition was not observed. Therefore, the LOELR was 3.2 mg/L, and the EL50 was >100 mg/L.

The study demonstrates was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice standards. The test substance could not be detected in any samples. However, the fact that fluorescence was statistically significantly inhibited in test chambers containing undiluted WAFs (≥3.2 mg/L loading rate) suggests that at least a portion of the test substance was in solution. The correlation of fluorescence with biomass was not addressed in detail, and the effect of the test substance on fluorescence was not controled. Therefore, this study is classified as acceptable with restrictions.