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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 11, 2017 to January 30, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Organization for Economic Cooperation and Development. 2011. OECD Guidelines for the
Testing of Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test.
Adopted 23 March 2006. Revised 28 July 2011.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Official Journal of the European Communities. 1992. No. L383 A. Method C.3. Algal Inhibition Test.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution into the test chambers and prior to inoculation. At exposure termination, samples were collected from the pooled replicates from each treatment and control group. At each sampling interval, 9.0 mL of test solution was collected from mid-depth and transferred into glass scintillation vials containing 1.0 mL of tetrahydrofuran. At each sampling interval, an additional set of samples was collected and stored under refrigerated conditions for possible future analysis.
Vehicle:
no
Details on test solutions:
A primary stock was prepared by dissolving 0.0450 g of the test substance in 1000 mL of freshwater AAP medium to achieve a nominal concentration of 45 mg/L. The primary stock was inverted at least 20 times, placed in an ultrasonic water bath for 88 minutes, and stirred overnight (17 hours and 44 minutes) prior to use. On the following day, the primary stock was placed in an ultrasonic water bath for 10 minutes and stirred while all subsequent dilutions were made. The primary stock appeared slightly cloudy with particulates on the surface.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The freshwater alga, Raphidocelis subcapitata, was selected as the test species for this study. The species is representative of an important group of freshwater algae, and was selected for use in the test based upon a past history of use, and ease of culturing in the laboratory. Original algal cultures were obtained from the University of Texas at Austin, and have been maintained in culture medium at EAG Laboratories, Easton, Maryland since June 2017. Algal cells used in this test were obtained from EAG Laboratories – Easton cultures that had been actively growing in culture medium under similar environmental conditions as used in this test for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh medium four days prior to test initiation.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No post exposure observation period specified.
Hardness:
Not specified
Test temperature:
Test chambers were held in an environmental chamber at a temperature of 24 ± 2ºC.
pH:
The pH of the medium was 7.5 ± 0.1.
Dissolved oxygen:
Not specified
Salinity:
Not applicable.
Conductivity:
Not specified
Nominal and measured concentrations:
Four test solutions were prepared at nominal concentrations of 2.8, 5.6, 11, 23 and 45 mg/L by diluting aliquots of the 45 mg/L primary stock with freshwater AAP medium.
Details on test conditions:
Environmental Conditions
Test chambers were held in an environmental chamber at a temperature of 24 ± 2ºC. The temperature of a container of water adjacent to the test chambers in the environmental chamber was measured continuously using an Amega Scientific Corporation centralized monitoring system. The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was 6,000 lux ± 10%. Light intensity was measured at test solution level at five locations surrounding the test flasks at test initiation using a SPER Scientific 840006C light meter. The pH of the medium in each treatment and control group was measured at test initiation and exposure termination using a Thermo Orion A214 pH meter. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and control group. At exposure termination, pH was measured in pooled samples of test solution collected from each of the remaining replicates of each treatment and control group.

Inoculation of Test Chambers
Prior to test initiation, the concentration of algal cells in the stock culture (culture identification No. 17-01) was determined using a hemacytometer and microscope, and was 3.39 x 106 cells/mL. In order to achieve the desired initial cell density of approximately 10,000 cells/mL, 0.295 mL of stock culture was added to each replicate test chamber at test initiation using an Eppendorf pipette.

Algal Growth Measurements and Observations
Test medium samples were collected from each replicate of the treatment and control groups for the determination of algal cell densities. Samples were collected at approximately 24-hour intervals during the 72-hour exposure and were held for a maximum of two days under refrigerated conditions sufficient to inhibit growth until cell counts could be performed. Cell counts were performed using an electronic particle counter (Coulter Electronics, Inc.). Prior to conducting cell counts, the linearity of the instrument response was determined at settings previously established for Raphidocelis subcapitata. A primary counting standard containing Raphidocelis subcapitata cells was prepared at a target nominal concentration of 100,000 cells/mL. The density of the primary counting standard was verified using a hemacytometer and a microscope, and the standard was subsequently diluted to provide a series of seven counting standards for the determination of instrument linearity. Theoretical densities were assigned to each secondary counting standard based upon the verified density of the primary counting standard and the dilution ratio. The cell densities of the counting standards were measured using the electronic particle counter and were compared to the theoretical densities by performing a least squares regression analysis. Cell counts for samples collected during the test were conducted once instrument linearity was demonstrated (i.e., the R-squared value obtained through the regression analysis was 0.99727). A single aliquot of each sample collected during the test was diluted with an electrolyte solution (Isoton®). Three, 0.5-mL volumes of the diluted sample were counted, and the resulting counts were averaged. The cell density of the sample was determined by adjusting the mean cell count (cells/mL) obtained using the particle counter, based upon the Y-intercept and slope calculated through the regression analysis, and the dilution factor.
Samples of test solution were collected from each replicate at the end of the test. These samples were pooled within their respective treatments, and sub-samples were removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Cells in the replicate test chambers also were assessed for aggregation or flocculation of cells, and adherence of the cells to the test chamber.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: cell density, growth rate, and yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: cell density, growth rate, and yield
Details on results:
Measurement of Test Concentrations
Results of analyses to measure concentrations of Tetrabromophthalic Anhydride in test solution samples collected during the study are presented in Table 2. Measured concentrations of Tetrabromophthalic Anhydride on day 0 in the treatment groups ranged from 69.1 to 78.3% of nominal. The additional set of samples collected on day 0 were analyzed for further evaluation. The results from the back-up samples confirmed the initial result. Measured concentrations of Tetrabromophthalic Anhydride at test termination in the treatment groups ranged from 99.2 to 108% of nominal. Mean measured concentrations were determined to be 2.5, 4.9, 9.4, 20, and 41 mg/L, equivalent to 88.8, 86.8, 85.5, 85.7, and 91.1% of nominal, respectively.

Observations and Measurements
Temperatures remained within the 24 ± 2°C range established for the test. The pH of all test solutions at test initiation ranged from 6.5 to 7.4, with pH decreasing with increasing test substance concentration. At test termination, pH measurements in samples of test solution obtained by pooling the replicates of each experimental group ranged from 8.1 to 9.4. The light intensity ranged from 5,640 to 6,560 lux, which was within the desired range of 6,000 lux ± 10%.
The toxicity of Tetrabromophthalic Anhydride to R.subcapitata was determined by evaluating changes in cell density over a 72-hour exposure period. Cell densities were used to calculate growth rates for each 24-hour interval of exposure and yields at 72 hours of exposure.

After 72 hours of exposure, inhibition of cell density in the 2.5, 4.9, 9.4, 20, and 41 mg/L treatment groups (based on mean measured concentrations) was -8, -6, -3, 7, and 9%, respectively, relative to the negative control. Inhibition of growth rate in the 2.5, 4.9, 9.4, 20, and 41 mg/L treatment groups was -1, -1, -1, 1, and 2%, respectively, relative to the negative control. Inhibition of yield in the 2.5, 4.9, 9.4, 20, and 41 mg/L treatment groups was -8, -6, -3, 7, and 9%, respectively, relative to the negative control. . The 72-hour NOEC was determined to be 41 mg/L. The 72-hour EC50 values for cell density, growth rate, and yield were determined to be >41 mg/L, for all three endpoints.
After 72 hours of exposure, adherence of cells to the test chambers was not observed in any of the control or treatment groups. Flocculation or aggregation of cells was not observed in any of the experimental groups. Cells present in all treatment groups appeared normal when compared to cells in the negative control.

Conditions for the Validity of the Test
Mean cell density in the control flasks increased by a factor of 251 after three days, achieving the 16X growth criterion. The coefficient of variation of average specific growth rates in the control replicates was 1.2%, achieving the less than 7% criterion. The mean percent coefficient of variation for section-by-section specific growth rates in the control replicates was 7.7%, achieving the less than 35% criterion.
Results with reference substance (positive control):
Not specified
Reported statistics and error estimates:
Dunnett’s test indicated mean cell density, mean growth rate, and mean yield were not significantly reduced (p > 0.05) in any of the Tetrabromophthalic Anhydride treatment groups when compared to the negative control

Temperature Measurements

Time (Days)

Temperature (°C)1

Mean

Low

High

0

1

2

3

24.25

24.23

24.21

24.37

24.07

24.13

24.07

24.32

24.32

24.32

24.38

24.38

0-3

24.27

24.07

24.38

1Temperature was continuously monitored in a container of water located adjacent to the study with an Amegaview Scientific centralized monitoring system.

 

pH Measurements

Mean Measured Test Concentration (mg/L)

pH Measurements

Day 01

Day 32

Negative Control

2.5

4.9

9.4

20

41

7.4

7.3

7.3

7.2

7.1

6.5

9.4

8.7

9.0

8.2

8.1

8.1

1Day 0 samples were collected from the individual batches of test solutions prepared for each treatment and control group at test initiation.

2Day 3 samples were from pooled samples of test solution collected from the three replicates per treatment and six replicates in the control group at test termination.

 

Inhibition of R. subcapitata Growth After 72 Hours of Exposure to Tetrabromophthalic Anhydride

72 Hours of Exposure1

Mean Measured Test Concentration (mg/L)

Mean Cell Density (cell/mL)3*

Percent Inhibition2

Mean Growth Rate (hours-1)*

Percent Inhibition2

Mean Yield (cells/mL)3*

Percent Inhibition2

Negative Control

2.5

4.9

9.4

20

41

2,506,690

2,696,749

2,648,854

2,575,488

2,341,068

2,290,691

--

-8

-6

-3

7

9

0.0767

0.0777

0.0775

0.0771

0.0757

0.0754

--

-1

-1

-1

1

2

2,296,690

2,686,749

2,638,854

2,565,488

2,331,068

2,280,691

--

-8

-6

-3

7

9

1Calculations were performed using SAS Version 9.4. Manual calculations may differ slightly.

2Inhibition was calculated relative to the mean negative control response.

3Yield was calculated as the final cell density minus the nominal initial cell density (10,000 cells/mL).

*None of the treatment group means were significantly different from the negative control mean (Dunnett’s test, P>0.05)

 

Validity criteria fulfilled:
yes
Conclusions:
The freshwater alga, Raphidocelis subcapitata, was exposed to a series of five treatment levels of Tetrabromophthalic Anhydride ranging from 2.5 to 41 mg/L, based on mean measured concentrations. The toxicity of Tetrabromophthalic Anhydride to Raphidocelis subcapitata was assessed based on effects on cell density, growth rate, and yield relative to the negative control. The 72-hour NOEC was determined to be 41 mg/L based on a lack of statistically significant reductions in cell density, growth rate, and yield in any of the treatment groups. The 72-hour EC10 values for cell density, growth rate, and yield were determined to be 32, >41, and 32 mg/L, respectively. The 72-hour EC50 values for cell density, growth rate, and yield were determined to be >41 mg/L, for all three endpoints.
Executive summary:

The objective of the study was to determine the toxicity of Tetrabromophthalic Anhydride, to the freshwater alga, Raphidocelis subcapitata, over a 72-hour exposure period.

 

The study was based on procedures outlined in OECD Guidelines for Testing of Chemicals, Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test; and the Official Journal of the European Communities No. L383 A, Method C.3.: Algal Inhibition Test.

 

The freshwater alga, Raphidocelis subcapitata, was exposed to a series of five treatment levels of Tetrabromophthalic Anhydride ranging from 2.5 to 41 mg/L, based on mean measured concentrations. The toxicity of Tetrabromophthalic Anhydride to Raphidocelis subcapitata was assessed based on effects on cell density, growth rate, and yield relative to the negative control.

 

The 72-hour NOEC was determined to be 41 mg/L based on a lack of statistically significant reductions in cell density, growth rate, and yield in any of the treatment groups. The 72-hour EC10 values for cell density, growth rate, and yield were determined to be 32, >41, and 32 mg/L, respectively. The 72-hour EC50 values for cell density, growth rate, and yield were determined to be >41 mg/L, for all three endpoints.

Description of key information

72-hour NOEC (cell density, growth rate and yield): 41 mg/L.

72-hour EC50 (cell density, growth rate, and yield) : >41 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
41 mg/L
EC10 or NOEC for freshwater algae:
41 mg/L

Additional information

The objective of the study was to determine the toxicity of Tetrabromophthalic Anhydride, to the freshwater alga, Raphidocelis subcapitata, over a 72-hour exposure period.

 

The freshwater alga, Raphidocelis subcapitata, was exposed to a series of five treatment levels of Tetrabromophthalic Anhydride ranging from 2.5 to 41 mg/L, based on mean measured concentrations. The toxicity of Tetrabromophthalic Anhydride to Raphidocelis subcapitata was assessed based on effects on cell density, growth rate, and yield relative to the negative control.

 

The 72-hour NOEC was determined to be 41 mg/L based on a lack of statistically significant reductions in cell density, growth rate, and yield in any of the treatment groups. The 72-hour EC10 values for cell density, growth rate, and yield were determined to be 32, >41, and 32 mg/L, respectively. The 72-hour EC50 values for cell density, growth rate, and yield were determined to be >41 mg/L, for all three endpoints.