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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)

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)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
The test solutions were prepared from a stock solution initially containing 100 mg of TNPP in 1 L of dilution water. TNPP is not water-soluble (< 0.6 µg/mL at 24 + 1 °C). The major hydrolysis product, nonylphenol, is also sparingly soluble in water. However, phosphorous acid released upon hydrolysis of TNPP is water-soluble. The substance was weighed on a glass Petri dish (100 mg) and the dish placed into a 2-L glass, Erlenmeyer flask containing 1 L of dilution water. A magnetic stir bar was added and the mouth of the flask sealed with Parafilm®. The test substance was stirred gently for 78 hours at room temperature (21 ± 2 °C). The test solutions were then prepared from the stock solution of TNPP hydrolysis products as recommended by the OECD for the testing of difficult substances. A 100-mL volume of the hydrolyzed stock solution was poured into a 250-mL plastic container for the highest test concentration (100 mg/L nominal test concentration). A second 100-mL volume of the stock solution was poured into another 250-mL container and serially diluted with 100-mL volumes of dilution water to obtain the remaining test concentrations (50.0, 25.0, 12.5, 6.3, 3.1, and 1.6 mg/L nominal test concentrations). The excess 100-mL volume was discarded. The solutions were spiked with 1 mL of a concentrated nutrient solution and then inoculated (1 mL) to give an initial cell density of 9,664 + 154 cells/mL. The inoculum was taken from an exponentially growing culture, washed twice with a sodium bicarbonate solution, and the cell number adjusted to give the desired initial cell density in the 100-mL test volume.

Test organisms

Test organisms (species):
other: other algae: Green algae (Raphidocelis subcapitata, formerly Selenastrum capricornutum)

Study design

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

Test conditions

pH:
The pH at test initiation and termination in the controls and 100.0 mg/L test solution ranged from 7.0 to 8.0. The pH was measured in one of the control wells and one well of the highest concentration at test initiation and termination with litmus paper (± 0.5 units).
Details on test conditions:
The test was conducted in a controlled environment chamber at 23 + 2 °C TEST SYSTEM- Test vessel: 96-well microplates (Costar®, Corning Incorporated) each plate has 12 columns of 8 wells each - Type (delete if not applicable): open / closed - Material, size, headspace, fill volume: well volume was 300 mL, test volume was 150 mL; The outer wells were filled with the control solution. The inner 6 wells in each column were filled with the test solutions. - No. of vessels per control (replicates): There were three sets of controls on each plate. TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: dechlorinated City of Calgary tap water (charcoal filtered and aerated) spiked with nutrients.- Hardness of 198 mg CaCO3/L- pH of 7.6- Alkalinity: 146 mg CaCO3/L- Ca/mg ratio:- Conductivity: 446 ms/cm.OTHER TEST CONDITIONS- Photoperiod: continuous- Light intensity and quality: at the plate surface of 4,370 lux provided by cool white fluorescent lights. EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : - Determination of cell concentrations: Cell numbers were obtained from optical density measurements at 430 nm calibrated against particle and cell counts at test termination. Cell counts were done with a Coulter Counter Model ZBI® particle counter equipped with a 100 mm aperture. The solutions were osmotically adjusted prior to counting. The counts at test initiation were done on solutions osmotically adjusted by adding 0.2 mL of a 50 % sodium chloride solution to 20 mL of the inoculated test solution. At test termination, 0.5 mL was removed from six wells (equal volumes from each well) of the control, 12.5 mg/L, and 100.0 mg/L concentrations from each replicate plate. The solutions were osmotically adjusted for counting with the addition of 20 mL of a 0.5 % sodium chloride solution. Each solution was counted until successive counts were within 10 % of each other.- Other: Growth inhibition was assessed as the decrease in cell numbers relative to controls. OTHER- The plates were read and rotated to a different position under the light bank each day. Optical density measurements at 430 nm were taken at test initiation and at 24, 48, and 72 h with a MRX Microplate Reader (Dynatech Laboratories). Particle counts were made on the controls, the 12.5 mg/L, and 100.0 mg/L test solutions at 72 h. The counts were converted to cell densities with the factor 93.6 (dilution factor of 80 times 1.17, an empirical constant relating instrument counts to cell numbers). A conversion factor was then derived by dividing the cell density by the optical density at 430 nm corrected for the initial optical density reading at test initiation (background). The relationship for converting optical density readings at 430 nm to cell densities was cell density (cells/mL) = 12,845,000 (optical density @ 430 nm at 24, 48, or 72 h minus the reading at test initiation). The optical density readings of the six replicate wells per concentration per plate were averaged and the averages converted into cell densities. The three columns of six control wells on each plate were averaged into a single value for derivation of the toxicity values. The toxicity values for the inhibiting effects of hydrolysis products on growth of Raphidocelis subcapitata were derived from the areas under the growth curves. The percent inhibition of cell growth at each test substance concentration was calculated as the difference between the area under the control curve and the area under the growth curve of each test substance concentration. The results from each replicate plate were treated separately for derivation of the toxicity values (three replicates).
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
meas. (not specified)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (not specified)
Basis for effect:
growth rate
Details on results:
- Exponential growth: The initial and final control cell densities were 9,664 cells/mL and 404,000 cells/mL, respectively. This was a 42-fold increase in cell density over the 72-h test period. A 16-fold increase was required for a valid test. - The LOEC as well as the 24, 48 and 72 h EC50 values were >100 mg/l. The NOEC was the highest concentration tested of 100 mg/l. The level of nonylphenol present in the test solutions under the conditions in which the stock solution was prepared, diluted, and tested was not toxic to unicellular green alga, Raphidocelis subcapitata.

Any other information on results incl. tables

The criterion for effect was growth inhibition based on a decrease in the area under the growth curves for each concentration relative to controls. The test medium contains 0.65 mg/L phosphate. Complete hydrolyses of the test substance (100 mg/L) would yield approximately 12 mg/L of phosphorous acid. The cell density in the highest test concentration at 72 h was 344 % greater than the controls. This represents approximately 1.5 additional doublings of the cell population exposed to the hydrolyzed TNPP solution when compared to the controls. The result indicates that hydrolysis of TNPP causes growth stimulation due to the liberation of phosphorous.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
No effect on aquatic plant life toxicity.
Executive summary:

There were no effects on algae up to 100 mg/L of TNPP (well in excess of the TNPP water solubility limit).