ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

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:

11 November 2019 to 14 November 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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical measurement was performed at the applied test concentration levels at the start of the test and 24-hour intervals thereafter during the experiment. Samples from the control were only taken at the start and at the end of the test.
Samples were taken from the test solutions and from the control solution. The test material samples were properly diluted into the calibrated range with diluent and analysed by an HPLC with UV detection method, based on the method validation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Because the test material was considered to be poorly soluble in water, test material stock solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test material solution (nominal loading rate of 100.0 mg/L) was prepared by dispersing/dissolving the amount of test material into the test medium (OECD Medium) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30 °C and then equilibrated for about 24 hours at approximately 20 °C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the appropriate 100 % saturated solution.
The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Culture conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.4 - 22.5 °C (flask); 20.9 - 22.8 °C (climate chamber)

pH:

7.19 - 8.62

Nominal and measured concentrations:

6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution (nominal)
0.022, 0.049, 0.125, 0.269 and 0.599 mg/L (measured geometric mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: Closed. Tthe flasks were covered with air-permeable stoppers.
- Fill volume: 100 mL
- Aeration: No. Flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension.
- Initial cells density: The test was started (0 hours) by inoculation of a biomass of approximately 10^4 algal cells per mL test medium.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes. Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
- Composition of reconstituted algal growth medium: Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.
Stock solution 1 (macro nutrients): NH4Cl (15.0 mg/L), MgCl2.6H2O (12.0 mg/L), CaCl2.2H2O (18.0 mg/L), MgSO4.7H2O (15.0 mg/L), KH2PO4 (1.6 mg/L)
Stock solution 2 (iron): FeCl3.6H2O (64.0 µg/L), Na2EDTA.2H2O (100.0 µg/L)
Stock solution 3 (trace elements): H3BO3 (185.0 µg/L), MnCl2.4H2O (415.0 µg/L), ZnCl2 (3.0 µg/L), CoCl2.6H2O (1.5 µg/L), CuCl2.2H2O (0.01 µg/L), Na2MoO4.2H2O (7.0 µg/L)
Stock solution 4 (bicarbonate): NaHCO3 (50.0 mg/L)

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous illumination.
- Light intensity and quality: Fluorescent lamps (with a spectral range of 400 - 700 nm) produced a light intensity of about 7421 lux (equivalent to ~100 µE/m²/s). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0, 0.1, 1, 10, 100 % saturated solution
- Results used to determine the conditions for the definitive study: Yes
A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test material plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
Because significant inhibition was observed at the highest concentration level (100.0 % saturated solution) during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one control were tested in the main experiment.
The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solutions. The biological results are based on measured geometric mean test material concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.599 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.125 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Morphological deviations of the algal cells
Thin, small/very small cells were observed at the examined concentrations of 25.0, 50.0 and 100.0 % saturated solutions (nominal) during the experiment.

Average specific growth rates
The results of the statistical evaluation (based on Bonferroni t-Test; a = 0.05) show that the 0 - 72 h average specific growth rate was statistically significantly different from the untreated control value at the tested concentration range of 0.125 – 0.599 mg/L (measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 0.049 mg/L (measured).
At the highest test material concentration of 0.599 mg/L only 36.5 % growth rate reduction (0 – 72 h) was observed. As such, the 72 h ErC50 was determined to be > 0.599 mg/L.
The 72 h ErC50 value was determined to be higher than 0.599 mg/L (measured), the highest level tested; 0.98 mg/L (95 % confidence limits: 0.64 – 0.49 mg/L) was calculated [by Probit analysis (TOXSTAT software)].

Areas under growth curves
The results of the statistical evaluation (based on Bonferroni t-Test; a = 0.05) show that the 0 - 72 h areas were statistically significantly different from the untreated control value at the tested concentration range of 0.125 - 0.599 mg/L (measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 0.049 mg/L (measured).
The 72 h EbC50 value was determined by Probit analysis as 0.25 mg/L (95 % confidence limits: 0.21 – 0.30 mg/L).

Yield
The results of the statistical evaluation (based on Bonferroni t-Test; a = 0.05) show that the 0 - 72 h yield was statistically significantly different from the untreated control value at the tested concentration range of 0.125 – 0.599 mg/L (measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 0.049 mg/L (measured).
The 72 h EyC50 value was determined by Probit analysis as 0.22 mg/L (95 % confidence limits: 0.19 – 0.26 mg/L).

Validity criteria
- The cell density in the control cultures increased by the factor of 71.17 within three days.
- The mean coefficient of variation for section-by-section specific growth rates (days 0 - 1; 1 - 2; 2 - 3) in the control cultures was 21.34 %.
- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.22 %.
All validity criteria were met; therefore, the study can be considered as valid.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 19/238-022AL) with the reference item potassium dichromate is (Batch Number: A0345704): 10 – 13 September 2019.
The 72h ErC 50: 0.82 mg/L, (95 % confidence limits: 0.76 – 0.90 mg/L)
The 72h EbC 50: 0.58 mg/L, (95 % confidence limits: 0.53 – 0.62 mg/L)
The 72h EyC 50: 0.49 mg/L, (95 % confidence limits: 0.46 – 0.54 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0 - 1, 1 - 2 and 2 - 3)) to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software.
The ErC10, EbC10, EyC10, ErC20, EbC20, EyC20, ErC50, EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test

Calculation of Exposure Concentrations

Nominal Concentration (% saturated solution)	Measured Concentrations (mg/L)				Geometric Mean
	0h/start	24h	48h*	72h/end	mg/L
Control	n.d.	n.d.	n.d.	n.d.	-
6.25	0.112*	0.027	0.006	0.012	0.022
12.5	0.262	0.068	0.014	0.024	0.049
25.0	0.520	0.138	0.041	0.082	0.125
50.0	1.075	0.288	0.095	0.179	0.269
100.0	2.171*	0.462	0.276	0.464	0.599

n.d. – not detected

* Note: the measured samples were outlier of the linearity range, however these concentrations were calculated with extrapolation because the deviation was not higher than 0.051 mg/L in case of the lowest concentration and the deviation was not higher than 0.0451 mg/L in case of the highest concentration.

Growth Rates (m) and Percentage Inhibition of µ During the Test Period

Concentration		Growth Rate (m) and % Inhibition ofm
Nominal [% saturated solution]	Measured [mg/L]	0–24 h		0–48 h		0–72 h
		m	%	m	%	m	%
Control	-	0.0549	0.0	0.0618	0.0	0.0592	0.0
6.25	0.022	0.0569	-3.6	0.0613	0.9	0.0588	0.8
12.5	0.049	0.0529	3.6	0.0613	0.9	0.0581	1.9
25.0	0.125	0.0498	9.3	0.0559*	9.6	0.0549*	7.4
50.0	0.269	0.0498	9.3	0.0441*	28.6	0.0469*	20.8
100.0	0.599	0.0458	16.6	0.0395*^+	36.1	0.0376*	36.5

*: Statistically significantly different compared to the control values (Bonferroni t-Test, a = 0.05).

⁺: At this value the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentration		Area Under the Growth Curves (A) and Percentage Inhibition of A
Nominal [% saturated solution]	Measured [mg/L]	0–24 h		0–48 h		0–72 h
		A	%	A	%	A	%
Control	-	34.0	0.0	290.0	0.0	1354.0	0.0
6.25	0.022	36.0	-5.9	288.0	0.7	1320.0	2.5
12.5	0.049	32.0	5.9	280.0	3.4	1272.0	6.1
25.0	0.125	28.0	17.6	220.0*	24.1	996.0*	26.4
50.0	0.269	28.0	17.6	144.0*	50.3	572.0*	57.8
100.0	0.599	24.0	29.4	116.0*	60.0	356.0*	73.7

*: Statistically significantly different compared to the control values (Bonferroni t-Test; a = 0.05).

Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration		Yield (Y) and % Inhibition of Y
Nominal [% saturated solution]	Measured [mg/L]	0–72 h
		Y	%
Control	-	70.2	0.0
6.25	0.022	68.0	3.1
12.5	0.049	64.7	7.8
25.0	0.125	51.0*	27.3
50.0	0.269	28.3*	59.6
100.0	0.599	14.3*	79.6

*: Statistically significantly different compared to the control values (Bonferroni t-Test; a = 0.05).

Summary of Biological Results

Parameter (0-72 h)	Growth Rate (r) [mg/L]		Yield (y) [mg/L]	Biomass (b) [mg/L]
Calculation based on measured geometric mean test material concentrations
EC10	0.14		0.05	0.06
95 % conf. limits	0.11 – 0.19		0.04 – 0.07	0.04 – 0.07
EC20	0.28		0.09	0.09
95 % conf. limits	0.22 – 0.35		0.07 – 0.10	0.08 – 0.11
EC50	> 0.599		0.22	0.25
95 % conf. limits	0.64 – 1.49		0.19 – 0.26	0.21 – 0.30
NOEC	0.049		0.049	0.049
LOEC	0.125		0.125	0.125

Measured Concentrations of the Test Material with the Confidence Interval (95 %)

Nominal Concentration (% saturated solution)	Measured Concentrations at the Start (mg/L)	Measured Concentrations After 1 Day (mg/L)	Measured Concentrations After 2 Days* (mg/L)	Measured Concentrations at the End (mg/L)
Control	Not detected	Not detected	Not detected	Not detected
6.25	0.112 ± 0.0046**	0.027 ± 0.0108	0.006 ± 0.0012	0.012 ± 0.0014
12.5	0.262 ± 0.0121	0.068 ± 0.0086	0.014 ± 0.0299	0.024 ± 0.0189
25	0.520 ± 0.0181	0.138 ± 0.0598	0.041 ± 0.0299	0.082 ± 0.0573
50	1.075 ± 0.0143	0.288 ± 0.2077	0.095 ± 0.0528	0.179 ± 0.0873
100	2.171 ± 0.0774**	0.462 ± 0.2077	0.276 ± 0.0729	0.464 ± 0.0739

*Note that more attention is taken during the ecotoxicological evaluation of these results regarding the obtained little but higher slope of regression line on this measuring day.

** The measured samples were outlier of the linearity range however these concentrations were calculated with extrapolation because the deviation was not higher than 0.051 mg/L in case of the lowest concentration and the deviation was not higher than 0.0451 mg/L in case of the highest concentration.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 72-hour ErC50 was determined to be > 0.599 mg/L (95 % confidence limits 0.64 - 1.49 mg/L). The 72-hour ErC10 and NOEC (growth rate) were determined to be 0.14 mg/L (95 % confidence limits 0.11 - 0.19 mg/L) and 0.049 mg/L, respectively.

Executive summary:

The effect of the test material on the growth of the unicellular green algal species, Pseudokirchneriella subcapitata, was assessed in a study which was conducted in accordance with the standardised guidelines OECD 201, EU Method C.3 and EPA OPPTS 850.5400, under GLP conditions over an exposure period of 72 h..

As a significant toxic response was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were tested in the main experiment.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solutions.

Test concentrations were analytically determined at the start of the test and 24-hour intervals thereafter during the experiment. The corresponding measured geometric mean test material concentrations were: 0.022, 0.049, 0.125, 0.269 and 0.599 mg/L.

The biological results are based on measured geometric mean test material concentrations.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a = 0.05) by TOXSTAT software.

The effect levels, and their confidence limits, were calculated using Probit analysis by TOXSTAT software.

Under the conditions of the study, the 72-hour ErC50 was determined to be > 0.599 mg/L (95 % confidence limits 0.64 - 1.49 mg/L). The 72-hour ErC10 and NOEC (growth rate) were determined to be 0.14 mg/L (95 % confidence limits 0.11 - 0.19 mg/L) and 0.049 mg/L, respectively.  

Description of key information

The effect of the test material on the growth of the unicellular green algal species, Pseudokirchneriella subcapitata, was assessed in a study which was conducted in accordance with the standardised guidelines OECD 201, EU Method C.3 and EPA OPPTS 850.5400, under GLP conditions over an exposure period of 72 h.

 

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solutions. Test concentrations were analytically determined at the start of the test and 24-hour intervals thereafter during the experiment. The corresponding measured geometric mean test material concentrations were: 0.022, 0.049, 0.125, 0.269 and 0.599 mg/L.

 

Under the conditions of the study, the 72-hour ErC50 was determined to be > 0.599 mg/L (95 % confidence limits 0.64 - 1.49 mg/L). The 72-hour ErC10 and NOEC (growth rate) were determined to be 0.14 mg/L (95 % confidence limits 0.11 - 0.19 mg/L) and 0.049 mg/L, respectively.  

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.049 mg/L