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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:
06-06-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Triphenyl phosphate 43%
tbuthyltriphenyl phosphate 57%

fits the material as described in Chapter 1.2
Analytical monitoring:
yes
Details on sampling:
The concentration of Triphenyl Phosphate active ingredient was measured in test solution samples collected at initiation (0 hour) and termination (72 hours) of the definitive test. Time 0 hour samples were collected from parent solutions. Time 72-hour samples were collected after combining replicate solutions by treatment.
At each sampling point, a 5-mL sample was collected from the control and each test substance treatment, placed in separate 10-mL culture tubes. The samples were diluted with 5 mL of ACN, and further diluted, if necessary, with 50:50 ACN:FWAM to provide final sample concentrations that fell within the analytical standard concentrations range (i.e., 0.18 to 5.0 µg/L).
Vehicle:
no
Details on test solutions:
A definitive test with a nominal concentration range of 0 (control), 0.0030, 0.0095, 0.031, 0.098, 0.31, and 1.0 mg a.i./L (equivalent to 0.0070, 0.022, 0.072, 0.23, 0.72, and 2.3 mg Phosflex 71B/L) was conducted. Transferring 0.0024 g of Phosflex 71B to a 1,000 mL glass volumetric flask, and bringing the flask to volume with test medium. This primary standard was inverted to mix, sonicated for approximately 30 minutes, and stirred for approximately 30 minutes on a magnetic stir plate with a teflon stir bar following preparation. Appropriate aliquots of the primary standard solution were diluted with test medium to a volume of 0.5 L to prepare the test substance treatments at concentrations of 0.0030, 0.0095, 0.031, 0.098, and 0.31 mg a.i./L.
Test Solution Temperature: 22.8 to 25.0°C
Test Solution pH: 7.4 to 7.5
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The parent stock of Pseudokirchneriella subcapitata, formerly known as Selenastrum capricornutum, was obtained from the Department of Botany, Culture Collection of Algae, University of Texas at Austin, on 18 July 2017. The parent stock was identified as Selenastrum capricornutum. The prepared cultures were maintained in a temperature-controlled environmental chamber under continuous light. Periodically, new cultures were cloned from an existing culture derived from the parent stock. All cultures were maintained under the same conditions as those used for testing. The algal culture used for this test was four days old and the biomass had increased exponentially (i.e., specific growth rate of 1.2 day-1) during the culture period.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.8 to 25.0°C
pH:
7.4 to 7.5
Nominal and measured concentrations:
Nominal Concentration (mg /L) - 0.0030, 0.0030 Abiotic, 0.0095, 0.031, 0.098, 0.31, 1.0
0 Hour - 0.0029 (96), 0.0029 (96), 0.0067 (71), 0.022 (71), 0.070 (71), 0.24 (79), 0.78 (78)
72 Hour - 0.0023 (76), 0.0027 (89), 0.0063 (66), 0.021 (67), 0.064 (65), 0.25 (81), 0.72 (72)


Details on test conditions:
Test Solution Temperature, measured at 0 and 72 hours:22.8 to 25.0°C. The temperature of the test solutions for the control and each test substance treatment level remained within the protocol specified temperature range of 24°C ± 2°C for the duration of the study.
Test Solution pH:7.4 to 7.5
Photoperiod: continuous light
Light Intensity:7,305 to 7,406 lux
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
Results based on geometric mean calculated Phosflex 71B from TPP
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Results based on geometric mean calculated Phosflex 71B from TPP
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Remarks on result:
other:
Remarks:
Results based on geometric mean calculated Phosflex 71B from TPP
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.05 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
Results based on geometric mean calculated Phosflex 71B from TPP
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Results based on geometric mean calculated Phosflex 71B from TPP
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.05 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Remarks on result:
other:
Remarks:
Results based on geometric mean calculated Phosflex 71B from TPP
Details on results:
Calculations of Triphenyl Phosphate concentrations in freshwater test solution samples were performed by the external standard analysis function of Analyst 1.6.2 software. A linear standard curve was derived by plotting concentration vs. response for calibration standards using least squares regression with 1/x weighting.
The concentration of Triphenyl Phosphate from each sample was determined directly from the standard curve. Equation and detailed calculations are presented in the attached report

After 72 hours of exposure, mean cell density in the control was 41.3 x 10^4 cells/mL, or 72 times the initial nominal cell density. The mean cell density in the Phosflex 71B treatments at 72 hours ranged from a low of 19.1 x 10^4 cells/mL at the concentration of 0.75 mg a.i./L (1.7 mg Phosflex 71B/L) to a high of 46.2 x 10^4 cells/mL at the concentration of 0.021 mg a.i./L (0.050 mg Phosflex 71B/L). Percent inhibition in algal growth at 72 hours, as compared to the control, ranged from 12% at the concentration of 0.021 mg a.i./L (0.050 mg Phosflex 71B/L) to 54% at the concentration of 0.75 mg a.i./L (1.7 mg Phosflex 71B/L).
Percent inhibition in area under the growth curve at 72 hours, as compared to the control, ranged from -5% at the concentration of 0.021 mg a.i./L (0.050 mg Phosflex 71B/L) to 48% at the concentration of 0.75 mg a.i./L (1.7 mg Phosflex 71B/L). The NOEC at 72 hours was 0.021 mg a.i./L based on geometric mean measured Triphenyl Phosphate active ingredient concentrations (0.050 mg Phosflex 71B/L, based on geometric mean calculated Phosflex 71B concentrations) and the lack of a statistically significant reduction in area under the growth curve at this and all lower test substance treatments. Based on geometric mean measured Triphenyl Phosphate active ingredient concentrations (geometric mean calculated Phosflex 71B/L concentrations) and area under the growth curve values, the estimated 72 hour EbC50 value was >0.75 mg a.i./L, the highest concentration tested (1.7 mg Phosflex 71B/L).
Percent inhibition in growth rate at 72 hours as compared to the control ranged from -3% at the concentration of 0.021 mg a.i./L (0.050 mg Phosflex 71B/L) to 18% at the concentration of 0.75 mg a.i./L (1.7 mg Phosflex 71B/L) (Figure 13 and Figure 14). The NOEC at 72 hours was 0.067 mg a.i./L, based on geometric mean measured Triphenyl Phosphate active ingredient concentrations (0.16 mg Phosflex 71B/L, based on geometric mean calculated Phosflex 71B concentrations) and the lack of a statistically significant reduction in growth rate from time zero at this and all lower test substance treatments. Based on growth rate and geometric mean measured Triphenyl Phosphate active ingredient concentrations (geometric mean calculated Phosflex 71B/L concentrations), the estimated 72 hour ErC50 was >0.75 mg a.i./L, the highest concentration tested (1.7 mg Phosflex 71B/L). The coefficient of variation of average specific growth rates during the whole test period in control replicates was 2%. The mean coefficient of variation in growth rate between adjacent time periods was 11% for the control replicates.
The percent inhibition in yield at 72 hours, as compared to the control, ranged from -12% at the concentration of 0.021 mg a.i./L (0.050 mg Phosflex 71B/L) to 54% at the concentration of 0.75 mg a.i./L (1.7 mg Phosflex 71B/L). The NOEC at 72 hours was 0.067 mg a.i./L, based on geometric mean measured Triphenyl Phosphate active ingredient concentrations (0.16 mg Phosflex 71B/L, based on geometric mean calculated Phosflex 71B concentrations) and the lack of a statistically significant reduction in yield at this, and all lower test substance treatments. Based on yield and geometric mean measured Triphenyl Phosphate active ingredient concentrations (geometric mean calculated Phosflex 71B/L concentrations), the estimated 72 hour EyC50 value was 0.64 mg a.i./L, with 95% confidence limits of 0.55 and 0.73 mg a.i./L (1.4 mg Phosflex 71B/L, with 95% confidence limits: of 1.3 and 1.6 mg Phosflex 71B/L).

The definitive test, as performed, was considered to be slightly above solubility in the FWAM test media.  The information provided by the client indicated that the water solubility was approximately 1 mg/L; however, recoveries, while slightly outside the 20% of nominals, gave indication of the test substance solubility in FWAM as approximately 0.7 mg/L, based on the analytical recoveries of the primary stock of 1.0 mg a.i./L at 0 hour. Given the unknown actual solubility of the test material in FWAM, these values were considered acceptable.

Results Based on Geometric Mean Measured Triphenyl Phosphate Active Ingredient Concentrations:

Hour

EC

Type

EC Value

(mg a.i./L)

95% Confidence Limits

(mg a.i./L)

NOEC

(mg a.i./L)

24 Hours

EbC50

>0.75

Not calculated

>0.75

ErC50

>0.75

Not calculated

>0.75

EyC50

>0.75

Not calculated

>0.75

48 Hours

EbC50

>0.75

0.29 – 4.7

0.021

ErC50

>0.75

Not calculated

0.021

EyC50

>0.75

0.40 – 1.9

0.021

72 Hours

EbC50

>0.75

0.65 – 0.94

0.021

ErC50

>0.75

Not calculated

0.067

EyC50

0.64

0.55 – 0.73

0.067

Note: EbCx= area under the growth curve, ErCx= growth rate from time zero, EyCx= yield

Results Based on Geometric Mean Calculated Phosflex 71B Concentrations:

Hour

EC

Type

EC Value

(mg Phosflex 71B/L)

95% Confidence Limits

(mgPhosflex 71B/L)

NOEC (mg Phosflex 71B/L)

24 Hours

EbC50

>1.7

Not calculated

>1.7

ErC50

>1.7

Not calculated

>1.7

EyC50

>1.7

Not calculated

>1.7

48 Hours

EbC50

>1.7

0.69 – 11

0.050

ErC50

>1.7

Not calculated

0.050

EyC50

>1.7

0.90 – 4.3

0.050

72 Hours

EbC50

>1.7

1.5 – 2.1

0.050

ErC50

>1.7

Not calculated

0.16

EyC50

1.4

1.3 – 1.6

0.16

Note: EbCx= area under the growth curve, ErCx= growth rate from time zero, EyCx= yield

Validity criteria fulfilled:
yes
Remarks:
The test acceptability criteria were met for this study.
Conclusions:
The number of algal cells in the control at test termination was greater than 16 times the number initially inoculated to verify logarithmic phase growth. The overall mean coefficient of variation for daily growth rates in the control replicates during the course of the test did not exceed 35%. The coefficient of variation of average specific growth rates during the whole test period in control replicates did not exceed 7%. The pH in the control did not increase more than 1.5 units during the study. This study satisfies the OECD guideline requirement for a growth inhibition test with Pseudokirchneriella subcapitata.

Executive summary:

Algae study performed for phosflex 71B. EC50 and NOEC values were calculated for the whole substance based on measured TPP concentrations. EC50 yield value 1.4 mg/L and NOEC value of 0.05 mg/L were calculated.

Description of key information

Three studies are available. The study Phosflex 71B: Growth Inhibition Test with the Unicellular Green Alga, (2017) was done

under OECD 201 guideline.

Phosflex 71B: Growth Inhibition Test with the Unicellular Green Alga,Pseudokirchneriella subcapitata


Key value for chemical safety assessment

EC50 for freshwater algae:
1.4 mg/L
EC10 or NOEC for freshwater algae:
0.05 mg/L

Additional information