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:
2000-03-21 to 2000-03-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Concentration of the DOTP sample: 97.5 % (area) by GC/FID
Storage conditions of test material: No data available
Aqueous Solubility: 0.35 mg/L in well water at 25 °C; 1.5 mg/L in deionized water at 25 °C.
Lot/Batch No.: 9020344
Physical appearance: clear, colorless liquid
Structure: confirmed using GC/MS

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentrations of test substance in the exposure solutions were verified at test start and after 24, 48, and 72 h of exposure. At 24, 48, and 72 h, aliquots of each of the test solutions and controls were centrifuged prior to analysis and the supernatants were assayed. A second aliquot of the light and dark control test solutions (without algae) were analyzed without centrifugation.

Test solutions

Vehicle:
yes
Details on test solutions:
STOCK SOLUTION
A stock solution was prepared by dilution 0.0501 g of the test substance to 2.522 g with N,N-dimethylformamide (DMF). The resulting concentration of the stock solution was 19.9 mg/g.

TEST SOLUTION
Using a sterile acid-washed 1-L Erlenmeyer flask containing a magnetic stir bar (10 mm x 80 mm) and 600.0 g sterile algal media, 63.6 µL of the stock solution were slowly added using a 100-µL gas tight syringe while stirring rapidly. The solution was allowed to mix for an additional 2 min. before removal of the time 0 aliquots for HPLC analysis.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
Common name: Green algae
Supplier: American Type Culture Collection, Rockville, MD
Age at study initiation: 4-day culture, passage 2 in liquid medium

Study design

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

Test conditions

Hardness:
No data
Test temperature:
24 °C
pH:
Negative controls: 7.45-7.47
Test flasks: 7.34-7.57
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal Concentration Tested: 2.0 mg/L
Measured Concentration: 0.86 mg/L
Details on test conditions:
Replicates: 3
Algal cell concentration: At test start, 333 µL of algal suspension were added to each flask to achieve a cell concentration of 1x10E4 cells/mL
Exposure vessels: sterile glass 250-mL Erlenmeyer flasks secured with sterile foam stoppers
Exposure solution volume: 100 mL
Incubator conditions: Shaking at 100 rpm, mean illumination of 742.3 ± 5.0 foot candles.
Test Parameters: Biomass and growth rate
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: There was no inhibition of growth of algae exposed to the test substance. Therefore, statistical analysis of the data was not performed.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: There was no inhibition of growth of algae exposed to the test substance. Therefore, statistical analysis of the data was not performed.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: There was no inhibition of growth of algae exposed to the test substance. Therefore, statistical analysis of the data was not performed.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.86 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: There was no inhibition of growth of algae exposed to the test substance. Therefore, statistical analysis of the data was not performed.
Details on results:
Measurements of cell growth were performed at times 24, 48, and 72 h. Inhibition of growth was not measured in any of the vessels during the study period.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The measured cell concentrations in the test cultures and controls were tabulated, together with the mean analyzed concentration of the test substance and the time of measurement. The mean values of the algal cell concentrations for the test substance concentration and for the controls were plotted against time to produce growth curves using EXCEL. To determine the concentration/effect relationship, two approaches were used: comparison of areas under the growth curves or biomass, and comparison of growth rates. Statistical analysis of the data was not necessary as inhibition in biomass or growth rate was not observed.

Any other information on results incl. tables

The stability of the test substance under the test conditions was determined by analyzing the exposure solutions at test start and at 24-h intervals through test end (72 h) using HPLC/DAD. At 24, 48, and 72 h, aliquots of all test vessels were analyzed following centrifugation. The light and dark chemical control vessels were also analyzed prior to centrifugation to determine loss of the test substance by adsorption to the algal cells and loss due to sample preparation. There was no significant loss of test substance when the chemical control flask was exposed to the incubator lighting conditions. The centrifugation step appeared to remove test substance from solution as there was 57.5% loss from the centrifuged light control flask compared to 2.4% loss from the light control that had not been centrifuged. Similar results were obtained when the dark chemical control flasks (centrifuged vs. non-centrifuged) were compared. The findings from the chemical controls indicate the aqueous solubility of the test substance had been exceeded and centrifugation removed the remaining undissolved test substance. The flasks containing the algae exposed to the test substance also exhibited loss of test substance concentration (71.9%). Based on the losses observed in the centrifuged flasks containing the chemical controls, most of the loss may be attributed to undissolved test substance. A small amount of test substance may have been adsorbed to the algal cells.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The mean cell concentration increased by a factor of 100-fold within 72 h. This meets the criteria of 16-fold increase required for a valid test.
Conclusions:
A saturated solution of DOTP is not expected to adversely affect aquatic plants.
Executive summary:

In a 72-h static toxicity study, unicellular green algae (Selenastrum capricornutum) were exposed to DOTP at a measured concentration of 0.86 mg/L under static conditions. No inhibition of biomass or growth rate was measured in this study. The 72-h EbC50 and ErC50 values were determined to be >0.86 mg/L. The 48-h NOEC value was >= 0.86 mg/L.