2-Propenoic acid, 2-hydroxyethyl ester, reaction products with O,O-bis(dialkyalkyl) hydrogen phosphorodithioate and dimethyl phosphonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 2019 to 06 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OCSPP Number 850.4500: Algal Toxicity

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Samples of the test solutions were collected at approximately 0, 24, 48, 72, and 96 hours to measure concentrations of total organic carbon (TOC).
- 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.
- Samples at 24, 48, and 72 hours were collected from a surrogate replicate included in each treatment and control group.
- At exposure termination, samples were collected from the pooled replicates from each treatment and control group.

Vehicle:

no

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

- The freshwater green 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 Culture Collection (UTEX), and had been maintained in culture medium at Eurofins, Easton, Maryland since June 2017.
- Algal cells used in the test were obtained from Eurofins-Easton cultures that had been actively growing in culture medium for at least two weeks prior to test initiation.
- Algal cells for the study were taken from a culture that had been transferred to fresh media four days prior to test initiation. The negative control organisms were expected to exhibit exponential growth over the 72-hour exposure period.
- The exponential growth phase, defined as the period of growth where the algal cells are dividing at a constant rate, is indicated by the linear section of the growth curve (see Figure 1, attached).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

Not reported

Test temperature:

24.33 to 25.45 °C

pH:

- 6.8 to 7.4 at test initiation (see Table 3, attached)
- 7.5 to 9.7 at test termination (see Table 3, attached)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal loading rates of 1.0, 3.0, 10, 30 and 100 mg test item

Details on test conditions:

OBJECTIVE
- The objective of this study was to determine the toxicity of the test item prepared as a water
accommodated fraction to the freshwater green alga (Raphidocelis subcapitata) during a 96-hour
exposure.

EXPERIMENTAL DESIGN
- The green alga (Raphidocelis subcapitata) was exposed to five test concentrations and a negative control (culture medium) under static conditions for 96 hours. Eight replicate test chambers in the control group and four replicate test chambers in all treatment groups except for the nominal 1.0 mg/L traeatment group were maintained. At test initiation, one of the replicates in the nominal 1.0 mg/L treatment group was inadvertently knocked over and consequently removed from the study. This deviation from the approved protocol was not considered to be detrimental to the study.
- Additional surrogate replicates were included in each experimental group to provide test solution for total organic carbon analysis at 24, 48, and 72 hours of exposure. Due to the low solubility of the test item, test solutions were prepared as water accommodated fractions (WAFs) and test concentrations are based upon the loading rate for each test solution. Nominal loading rates were selected in consultation with the Sponsor and were based upon results of exploratory, non-GLP range finding toxicity tests and preliminary analytical results.
- A range-finding test conducted with nominal WAF loading rates of 1.0, 3.0, 10, 30, and 100 mg/L, and yielded -1, 32, 30, 79, and 97 % inhibition of mean cell density after 96 hours of exposure, respectively, relative to the mean negative control response (Table 1). Nominal loading rates selected were 1.0, 3.0, 10, 30, and 100 mg/L. Samples of test medium were collected from each treatment and control group at approximately 0, 24, 48, 72, and 96 hours of exposure for total organic carbon (TOC) analysis.
- At test initiation an inoculum of the algal cells was added to each test chamber to achieve a nominal concentration of approximately 10,000 Raphidocelis cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities.
- Cell densities were used to determine area under the growth curve, growth rate and yield which were subsequently used to calculate percent inhibition values relative to the negative control over the 96-hour exposure period. EbLx, ErLx, and EyLx values (i.e., the theoretical loading rates that would produce an x % reduction in area under the growth curve, growth rate, and yield, respectively) were determined, when possible, at 72 and 96 hours of exposure. No-observed-effect-loading-rate (NOELR) were determined at 72 and 96 hours through statistical evaluation of the area under the growth curve, growth rate and yield data, as well as examination of the concentration-response pattern.

TEST APPARATUS
- Test chambers were sterile, 250-mL Erlenmeyer flasks plugged with foam stoppers and contained
100 mL of test or control medium.
- The test flasks were labelled with the project number, concentration and replicate, and were indiscriminately positioned daily on a mechanical shaker in an environmental chamber designed to maintain the desired test temperature throughout the test.
- The test flasks were shaken continuously at 100 rpm.

CULTURE MEDIUM
- The algal cells were cultured and tested in freshwater AAP medium.
- Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified Eurofins EAG Agroscience LLC well water.
- The test medium then was prepared by adding appropriate volumes of the stock nutrient solutions
to purified well water (see Appendix 3, attached).
- The pH was adjusted to 7.5 ± 0.1 using 10 % hydrochloric acid and 0.1 N sodium hydroxide, as needed, and the medium was sterilised by filtration (0.22 µm) prior to use.
- Results of the most recent analyses performed to measure the concentrations of selected contaminants in the well water used to prepare the algal medium are presented in Appendix 4 (attached).

ENVIRONMENTAL CONDITIONS
- Test chambers were held in an environmental chamber at a temperature of 24 ± 2 °C.
- The temperature of a container of water adjacent to the test chambers in the environmental chamber was measured continuously using a Pointview Scientific Corporation centralised monitoring system.
- The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was 4,300 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 Model A215 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.

PREPARATION OF TEST CONCENTRATIONS
- Individual WAF solutions were prepared by mixing a calculated amount of test substance in freshwater AAP medium at nominal loading rates of 1.0, 3.0, 10, 30, and 100 mg/L. Loading rates were not corrected for percent active ingredient in the test substance. The negative control solution consisted freshwater AAP medium without test substance added.
- Glass aspirator bottles were filled with 4 or 10 L of freshwater AAP medium measured with 2 or 4 L glass volumetric flasks. Required mass of test substance for each solution was weighed into tared plastic weigh boats using an analytical balance and directly added to each aspirator bottle. Water accommodated fractions were stirred with a Teflon-coated stirbar and magnetic stir plate overnight (20 hours and 54 minutes). While stirring, a vortex depth of 30% of the test solution height was maintained.
- After mixing overnight, all test solutions appeared clear and colourless; however, test material adhered to the walls of the aspirator bottles and particulates were suspended in the water column of all treatment levels. The test solutions were decanted from mid-depth via tubing on each aspirator bottle and filtered through a 0.22 micron nitrocellulose filter to remove any undissolved material. Test solutions were decanted and filtered from lowest to increasing test item concentration.

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 2.34 x 106 cells/mL.
- In order to achieve the desired initial cell density of approximately 10,000 cells/mL, 0.427 mL of stock culture was added to each replicate test chamber at test initiation using a Sartorius digital pipette.

ALGAL GROWTH MEASUREMENTS AND OBSERVATIONS
- Test medium samples were collected from each biological replicate of the treatment and the control groups for the determination of algal cell densities. Samples were collected at approximately
24-hour intervals during the 96-hour exposure and were held for a maximum of four days under
refrigerated conditions sufficient to inhibit growth until cell counts could be performed. Samples
collected on day 4 were recounted 10 days after collection to evaluate the original count data. 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 R. subcapitata cells was prepared, the density 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 r2 value obtained through the regression analysis was 0.99979). 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. The following equation was used: Cell density of sample (cells/mL) = [(Mean cell count (cells/mL) – y – intercept) / slope] * 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.

STATISTICAL ANALYSES
- The calculation of cell densities, area under the growth curve, yield and growth rates and percent
inhibition values, as well as all statistical analyses, were conducted using “The SAS System for Windows, Version 9.4".
- Growth rate was calculated for each replicate of the control and treatment groups at each 24-hour interval of exposure using the formula µ = (ln Nn – ln N0) / (tn – t0) where µ = average specific growth rate; N0 = nominal cell density (cells/mL) at t0; Nn = measured cell density (cells/mL) at tn; t0 = time of beginning of test (hours); tn = time after beginning of test (hours).
- Area under the growth curve (biomass) was calculated for each replicate of the control and
treatment groups at each 24-hour interval of exposure using the formula A = ((N1-N0)/2)(t1)+((N1+N2-2N0)/2)(t2-t1)+…+((Nn-1+Nn-2N0)/2)(tn-tn-1) where A = Area under the growth curve; N0 = Mean nominal number of cells/mL at t0; N1 = Mean measured number of cells/mL at t1; N2 = Mean measured number of cells/mL at t2; Nn = Mean measured number of cells/mL at tn; t0 = Time of beginning of test (hours); t1 = Time of first measurement after beginning of test (hours); t2 = Time of second measurement after beginning of test (hours); tn = Time of nth measurement after beginning of test (hours).
- Final population density (yield) was calculated for each replicate of the control and treatment groups as the final biomass (cell density) in the exposure period minus the initial biomass (nominal cell density) at the 0-72 and 0-96-hour intervals of exposure. Inhibition values were calculated for each treatment group as the percent reduction in area under the growth curve, growth rate and yield relative to the negative control replicates using the formula Percent inhibition = [(Mean control response – Mean treatment response) / Mean control response] * 100
- ELx values in this study represent the theoretical test loading rates that would produce an x % reduction in a variable of interest relative to the control. ELx values and their corresponding 95%
confidence intervals were estimated, when possible, using non-linear regression with treatment response (area under the growth curve, growth rate, and yield) and nominal WAF loading rates.
- The 72- and 96-hour area under the growth curve, growth rate and yield data were evaluated for normality and homogeneity of variance (α = 0.01) using Shapiro-Wilk’s and Levene’s tests, respectively. The 72 and 96-hour data met assumptions of normality or homogeneity of variance. The mean treatment group responses were compared to the negative control response using Dunnett’s one-tailed t-test (α = 0.05). NOELR values were based on the results of the statistical analyses as well as evaluation of the dose-response.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

biomass

Remarks on result:

other: 95 % confidence limits 33 to 45 mg/L

Remarks:

see full results summary (attached)

Key result

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits not applicable

Remarks:

see full results summary (attached)

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: yield

Remarks on result:

other: 95 % confidence limits 18 to 31 mg/L

Remarks:

see full results summary (attached)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

biomass

Remarks on result:

other: see full results summary (attached)

Key result

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Remarks on result:

other: see full results summary (attached)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

other: yield

Remarks on result:

other: see full results summary (attached)

Details on results:

MEASUREMENT OF TOTAL ORGANIC CARBON IN TEST SOLUTIONS
- Results of analyses for total organic carbon (TOC) in the test solutions are presented in Table 2 (attached).
- Measurements of TOC in the 1.0, 3.0, 10, 30, and 100 mg/L treatment groups at test initiation (0 hour) were < LOQ, < LOQ, < LOQ, 2.1, and 5.8 mg C/L. Measurements of TOC in samples collected from the pooled replicates of each test item treatment group at test termination (96 hours) ranged from 3.4 to 5.4 mg C/L, but no clear trend was observed. The low recoveries and the high variability of the results seen in the TOC results are attributed to the low carbon content in the test solutions. Since the concentrations of the test substance in the test solutions could not be monitored using results of the TOC analysis, the results of the study were based on the nominal test concentrations.
- The results of the study are based on nominal WAF loading rates. Water accommodated fractions are commonly used for UVCBs and the high masses of test substance added are not fully dissolved in the test medium, which was noted in the study by test material adhering to the walls of the aspirator bottles and observations of particulates suspended in the water column in all test item treatment groups. Nominal WAF loading rates were considered to be more applicable based on the concept of WAF studies.

OBSERVATIONS AND MEASUREMENTS
- Measurements of pH and light intensity are presented in Tables 3 and 4, respectively (attached).
- Temperature in a container of water located adjacent to the test ranged from 24.33 to 25.45 °C and remained within the 24 ± 2°C range established for the test.
- The pH of the test solutions at test initiation ranged from 6.8 to 7.4. At test termination, pH in the pooled replicates of each respective treatment and control group ranged from 7.5 to 9.7. The observed increase in pH is typical for tests conducted with R. subcapitata and is attributed to the photosynthetic activity of the algae. Although the pH in the control groups increased by greater than 1.5 units after 72 hours of exposure, the increases in pH documented for this study are commonly observed for this algal species and all control validity criteria were achieved despite the increase in pH.
- The light intensity ranged from 4,110 to 4,710 lux, which was within the desired range of 4,300 lux ± 10 %.
- The toxicity of the test item was determined by evaluating changes in cell density over a 96-hour exposure period. Cell densities were used to calculate area under the growth curve and growth rates for each 24-hour interval of exposure and yields at 72 and 96 hours of exposure. Inhibition of mean area under the growth curve, mean growth rate, and mean yield after 72 and 96 hours of exposure are presented in Tables 5 and 6, respectively (attached).
- The NOELR, EbLx, ErLx, and EyLx values and their corresponding 95 % confidence limits calculated using nominal WAF loading rates for area under the growth curve, growth rates, and yield, respectively, are presented in Table 7 (attached).
- Individual replicate data for each growth parameter, including cell densities, are presented in Appendices 6, 7, 8, and 9 (attached).
- Figure 1 (attached) illustrates changes in mean cell density in the negative control group throughout the test.
- Figure 2 (attached) depicts changes in mean cell density in all experimental cohorts over the 96-hour exposure period.
- Concentration-response curves for each parameter illustrating the dose-response response of the treatments and fit of the regression line to the data are presented in Figures 3 and 4 (attached).
- After 72 hours of exposure, inhibition of area under the growth curve in the 1.0, 3.0, 10, 30, and 100 mg/L treatment groups (based on nominal WAF loading rates) was 4, 0, 16, 40, and 87 %, respectively, relative to the negative control. Inhibition of growth rate in the 1.0, 3.0, 10, 30, and 100 mg/L treatment groups was 0, 0, 3, 10, and 46 %, respectively, relative to the negative control. Inhibition of yield in the 1.0, 3.0, 10, 30, and 100 mg/L treatment groups was 3, -1, 17, 43, and 92 %,
respectively, relative to the negative control. Based on nominal WAF loading rates, the 72-hour EbL50, ErL50, and EyL50 values were determined to be 37, >100, and 33 mg/L, respectively. Mean area under the growth curve, mean growth rate, and mean yield were significantly reduced (Dunnett’s test; p < 0.05) in the 30 and 100 mg/L treatment groups when compared to the negative control. The 72-hour NOELR was determined to be 10 mg/L.
- After 96 hours of exposure, inhibition of area under the growth curve in the 1.0, 3.0, 10, 30, and 100 mg/L groups (based on nominal WAF loading rates) was 16, 14, 23, 40, and 91 %, respectively, relative to the negative control. Inhibition of growth rate in the 1.0, 3.0, 10, 30, and 100 mg/L treatment groups was 4, 4, 4, 7, and 37%, respectively, relative to the negative control. Inhibition of yield in the 1.0, 3.0, 10, 30, and 100 mg/L treatment groups was 24, 22, 26, 39, and 92%, respectively, relative to the negative control. Based on mean measured concentrations, the 96-hour EbL50, ErL50, and EyL50 values were determined to be 39, >100, and 41 mg/L, respectively.
- Mean area under the growth curve, mean growth rate, and mean yield were significantly reduced based on the results of Dunnett’s test (p < 0.05) in all test item treatment groups when compared to the negative control. Although reductions in mean growth rate were determined to be statistically significant in the 1.0, 3.0, and 10 mg/L treatment groups, these reductions was not considered to be biologically significant. Reductions in area under the growth curve in the 1.0 and 3.0 mg/L treatment groups were also considered to be not biologically significant despite demonstrating statistical significance from the control response. Percent inhibition values for mean yield relative to the control in all test item treatment groups were considered to be biologically significant.
- The mean cell density for the control at 96 hours was approximately 4.0 x 10E+06 cells higher than is typically observed for toxicity tests conducted with R. subcapitata based on historical control data. Mean cell density values for the 1.0, 3.0, 10, and 30 mg/L treatment groups were also elevated based on historical control data. The samples collected at 96 hours of exposure were initially counted on 02 December 2019 and recounted by the Study Director on 06 December 2019 to evaluate the initial results and to ensure the high cell density values were not the result of an error.
- The results of the cell counts conducted on December 06, 2019 were reported, however, the counts were similar and confirmed the initial results. The environmental parameters for the
study were within the desired range and there was no apparent explanation for the increased growth response from 72-96 hours. The control data met all validity criteria and exponential growth was maintained over the 96-hour study. The 72-hour NOELR values for area under the growth curve and growth rate were determined based on evaluation of the dose-response rather than the statistical results. A 96-hour EyC05 was estimated to be 14 mg/L with a 95 % confidence interval of 9.5 to 20 mg/L and was reported in place of a NOELR. The 96-hour NOELR values for area under the growth curve and growth rate were determined to be 3.0 and 10 mg/L, respectively. The overall 96-hour NOELR was determined to be 3.0 mg/L based on area under the growth curve.
- After 96 hours of exposure, flocculation or aggregation of cells was not observed in the controls or any of the test item treatment groups. Adherence of cells to the test chambers was not observed in
any of the experimental groups. Cells in the 1.0, 3.0, 10, and 30 mg/L treatment groups appeared normal when compared to cells present in the negative control. Cells in the 100 mg/L treatment group appeared enlarged when compared to the negative control. No particulates or surface-slicks were observed in any of the treatment groups over the exposure period.

VALIDITY OF THE TEST
- Cell density increased exponentially in the negative control replicates over the 96-hour exposure period. Mean cell density increased by a factor of 237 after three days and a factor of 974 after four days.
- The coefficient of variation of average specific growth rates in the negative control replicates during the first 72 hours was 2.54 %.
- The mean percent coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the negative control replicates was 6.22 %.
- At test termination the coefficient of variation for mean growth rate and mean yield in the negative control replicates was 0.660 and 4.52 %, respectively (see Appendix 9, attached).

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

See above

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour values were determined to be 37 mg/L (EbL50), > 100 mg/L (ErL50) and 33 mg/L (EyL50). The 96-hour values were determined to be 39 mg/L (EbL50), > 100 mg/L (ErL50) and 41 mg/L (EyL50). The 72-hour NOELR values were reported as 10 mg/L based on biomass, growth rate and yield. The 96-hour NOELR value was reported as 3.0 mg/L based on biomass. The 96-hour NOELR value was reported as 10 mg/L based on growth rate. The 96-hour NOELR value was reported as < 1.0 mg/L based on yield.

Executive summary:

GUIDELINE

The study protocol was based on procedures outlined in the OECD Guideline for Testing of Chemicals, 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test; the Official Journal of the European Communities No. L 383 A, Method C.3. Algal Inhibition Test (2); and in the ASTM Standard E1218-04(2012) Standard Guide for Conducting Static Toxicity Tests with Microalgae.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item, the investigation was performed using a water accommodated fraction (WAF). The alga was exposed to nominal WAF loading rates of 1.0, 3.0, 10, 30 and 100 mg test item and a control (culture medium) for 96 hours under static renewal conditions. Samples were analysed for total organic carbon since

chromatographic methods for analysis were unobtainable due to the contributions in the test matrix.

 

RESULTS

Toxicity of the test item was assessed based on effects on area under the growth curve, growth rate and yield. All control validity criteria specified in the study protocol were achieved.

 

CONCLUSION

The 72-hour values were determined to be 37 mg/L (EbL50), > 100 mg/L (ErL50) and 33 mg/L (EyL50). The 96-hour values were determined to be 39 mg/L (EbL50), > 100 mg/L (ErL50) and 41 mg/L (EyL50). The 72-hour NOELR values were reported as 10 mg/L based on biomass, growth rate and yield. The 96-hour NOELR value was reported as 3.0 mg/L based on biomass. The 96-hour NOELR value was reported as 10 mg/L based on growth rate. The 96-hour NOELR value was reported as < 1.0 mg/L based on yield.

Description of key information

Based on nominal WAF loading rate, the EL50 (96 h) value for Raphidocelis subcapitata was determined to be > 100 mg/L based on growth rate. The NOELR (96 h) value was reported as 10 mg/L based on growth rate (OECD 201 and OCSPP 850.4500).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

GUIDELINE

The study protocol was based on procedures outlined in the OECD Guideline for Testing of Chemicals, 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test; the Official Journal of the European Communities No. L 383 A, Method C.3. Algal Inhibition Test (2); and in the ASTM Standard E1218-04(2012) Standard Guide for Conducting Static Toxicity Tests with Microalgae.

 

METHODS

Due to the low aqueous solubility and complex nature of the test item, the investigation was performed using a water accommodated fraction (WAF). The alga was exposed to nominal WAF loading rates of 1.0, 3.0, 10, 30 and 100 mg test item and a control (culture medium) for 96 hours under static renewal conditions. Samples were analysed for total organic carbon since

chromatographic methods for analysis were unobtainable due to the contributions in the test matrix.

 

RESULTS

Toxicity of the test item was assessed based on effects on area under the growth curve, growth rate and yield. All control validity criteria specified in the study protocol were achieved.

 

CONCLUSION

The 72-hour values were determined to be 37 mg/L (EbL50), > 100 mg/L (ErL50) and 33 mg/L (EyL50). The 96-hour values were determined to be 39 mg/L (EbL50), > 100 mg/L (ErL50) and 41 mg/L (EyL50). The 72-hour NOELR values were reported as 10 mg/L based on biomass, growth rate and yield. The 96-hour NOELR value was reported as 3.0 mg/L based on biomass. The 96-hour NOELR value was reported as 10 mg/L based on growth rate. The 96-hour NOELR value was reported as < 1.0 mg/L based on yield.