Registration Dossier

Ecotoxicological information

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:
2000-08-23 until 2001-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Butyl methoxydibenzoylmethan (BMDBM)
- Substance type: organic mono constituent
- Physical state: White to pale yellow powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 - 100 mg/l
- Sampling method: During the final limit test samples were taken from the filtrate prepared at 100 mg/i and the blank-control.
Sampling:
Frequency At t= 0 h, t = 24 h, and t= 96 h.
Volume 10 ml from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were analysed on the day of sampling for the final test.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation started with solutions containing a loading of 100 mg/I. These solutions were stirred for 3 days in the dark. The resulting suspensions were then filtered through a paper filter (5 µm) to remove the fraction of non-dissolved test substance particles. The lower test concentrationsfor the combined limit/range-finding test were prepared by subsequent dilutions of the stock in test medium. The final test solutions were all clear and colourless. The control solution received similar treatment to correct for possible influences of the treatment procedures. During preparation all solutions were as much as possible protected from light. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 10^4 cells/ml.
- Controls: Test medium without test substance or other additives (0 mg/I).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none reported

Test organisms

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): laboratory
- Age of inoculum (at test initiation): no data
- 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 (4000-9000 lux) in a climate room at a temperature of 23 ±2°C.


ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2x10^4 cells/mI.
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: none reported

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
24 mg/l as CaCO3 (= 0.24 mmol/l)
Test temperature:
23 ±2°C
pH:
At test start: 8.0 - 8.1
At test end: 9.2 - 9.4
Dissolved oxygen:
no data
Salinity:
Not particularly stated, however in the original study report a detailed description is given of the composition of M2-medium according to the ISO-Standard “Algal growth inhibition test”, formulated using Milli-Q water.
Details on test conditions:
Nominal concentration: 100 mg/l

Measured:
Sampling time: hours 0
Flitered sample measured concentration: 0.13 - 0.25 mg/l

Sampling time: hours 96
Flitered sample measured concentration: < 0.04 mg/l (= LOD) - 0.09 mg/l
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 0.055 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: 5 µm filtered test solution prepared at a nominal loading of 100 mg/I; max. water solubility determined to be 0.03 mg/L.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.055 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: 5 µm filtered test solution prepared at a nominal loading of 100 mg/I; max. water solubility determined to be 0.03 mg/l.
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF: 5 µm filtered test solution prepared at a nominal loading of 100 mg/L, the regulatory limit concentration.
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF: 5 µm filtered test solution prepared at a nominal loading of 100 mg/L, the regulatory limit concentration.
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none reported
- Any stimulation of growth found in any treatment: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: prevented as much as possible. Still, in all test solutions increasingly amounts of undissolved test material were present, probably in various amounts. The observed variation was therefore
probably mainly a consequence of testing above the solubility limit and perhaps partly related to biological parameters such as, for instance, variations in light intensity.
- Effect concentrations exceeding solubility of substance in test medium: Yes. Nominal concentration 100 mg/l. Water solubility reported to be 3 µg/l only.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: The EC50 for cell growth inhibition (EbC50:0-72h) was 0.59 mg/I with a 95 % confidence interval ranging from 0.32 to 1.1 mg/I.
Reported statistics and error estimates:
Chi-square test, F-test, ANOVA, Bioequivalence t-Test (P=0.05)

Any other information on results incl. tables

Table 2: Mean cell densities (x10^4 cells/ml) during the limit test

  Nominal conc. (mg/l)  0 h  24 h  48 h  72 h  96 h   
  1.0  1.0  3.1  30.6  132.3   
  100  1.0  1.1  3.5  25.5  104.1   

Filtrate prepared at a nominal loading of 100 mg/l.

Table 3: Percentage inhibition of cell growth during the limit test

  Nominal conc. (% filtrate)   Cell growth 0 -96h Cell growth 0 -96h 
    Mean area (A)  Mean area (B) 
  2336.74   
  100  1889.81  19.1 

Filtrate prepared at a nominal loading of 100 mg/l.

Algal cell growth was statistically significant inhibited according to the Bioequivalence t-Test (P=0.05). Cell growth in the six test vessels was inhibited by an average of 19.1% compared to the average cell growth in the controls. The individual inhibition per test vessel however ranged between 0 and 36%. Hence, a relatively large variation was found in total biomass. Note that the measured concentration decreased significantly during the test period, as a consequence of the low solubility, but remained more than a factor of 10 above the solubility limit of the test item in water. Thus, in all test solutions increasingly amounts of undissolved test material were present, probably iIn all test solutions increasingly amounts of undissolved test material were present, probably in various amounts. The observed variation was therefore probably mainly a consequence of testing above the solubility limit and perhaps partly related to biological parameters such as, for instance, variations in light intensity.

In conclusion, the apparent inhibition at a 5 µm filtered test solution prepared at a nominal loading of 100 mg/l is caused by variation in the extinction values and is therefore not considered to be biologically relevant.

Table 4: Percentage reduction of growth rate at various time intervals during the limit test

 Nominal conc. (% filtrate) Mean growth rate                   
   µ (24 -48h)  Reduction (%)   µ (24 -72h) Reduction (%)    µ (24 -96h)  Reduction (%)  
 0.04725  

 0.07123

  0.06782     
100   0.04824 -2.1  0.06560  8.0  0.06310  7.0   

Filtrate prepared at a nominal loading of 100 mg/l.

The growth rate was reduced by 7.0% during the 96-hour exposure period at a 5   µm filtered test solution prepared at a nominal loading of 100 mg/I. This was not biologically significant. It was also not statistically significant reduced according to the Bioequivalence t-Test (P=0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The actual exposure concentration could not be maintained above 80 % relative to the initial concentration. The mean measured concentration decreased significantly during the test period due to low solubility but remained above the water solubility.
Conclusions:
Under the conditions of the present study with Selenastrum capricornutum, butyl methoxydibenzoylmethane did not reduce growth rate of this fresh water algae species significantly at a 5 µm filtered test solution prepared at a nominal loading of 100 mg/I. The EC50 for both algal growth inhibition and growth rate reduction exceeded the maximum solubility of butyl methoxydibenzoylmethane in test medium at a 5 µm filtered test solution prepared at a nominal loading of 100 mg/l, the regulatory limit concentration.
Executive summary:

Selenastrum capricornutum, Fresh Water Algal Growth inhibition Test with butyl methoxydibenzoylmethane. The study procedures described in this report were based on OECD guideline No. 201. Under the conditions of the present study with Selenastrum capricornutum, butyl methoxydibenzoylmethane did not reduce growth rate of this fresh water algae species significantly at a 5 µm filtered test solution prepared at a nominal loading of 100 mg/l.

The test item was not soluble in test medium at 100 mg/I. The water solubility deteremined in water olutbility study was 0.03 mg/l according to data supplied by the sponsor. Analysis of the samples taken during the limit test showed that the measured concentration decreased from 0.145 mg/l at the start of the test to 0.053 mg/l after 24 hours and to 0.037 mg/l after 96 hours of exposure. The time weighted average concentration was determined to be 0.055 mg/l being the geometric mean of the concentrations measured during the test period.

Under the conditions of the present study with Selenastrum capricornutum, butyl methoxydibenzoylmethane did not reduce growth rate of this fresh water algae species significantly at a 5 µm filtered test solution prepared at a nominal loading of 100 mg/I. The EC50 for both algal growth inhibition and growth rate reduction exceeded the maximum solubility of butyl methoxydibenzoylmethane in test medium at a 5 µm filtered test solution prepared at a nominal loading of 100 mg/l, the regulatory limit concentration.