2-({[4-({bis[4-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)phenoxy](sulfanylidene)-$l^{5}-phosphanyl}oxy)phenyl]carbamoyl}oxy)ethyl prop-2-enoate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Water solubility: 0.01 mg/L
Stability of test concentration/s during exposure: Examined by chemical analysis (HPLC) at 0, 24, 48 and 72 hours.

Analytical monitoring:

yes

Remarks:

HPLC/MS analysis

Details on sampling:

Storage: Samples were diluted with acetonitrile (1:1; v/v) and then stored deep frozen and protected from light.

Vehicle:

yes

Details on test solutions:

PRE-TREATMENT OF TEST ITEM AND PREPARATION OF TEST ITEM CONCENTRATIONS
To produce the only test item concentration 200.6 mg of the test item were added to 2 litres of dilution water, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 - 12 µm. The pH was measured to be 7.8.
100 mL of the solution were taken and 0.564 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 6 replicates were prepared. All flasks were sealed with cotton stoppers.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Name: Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG
Source: Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 µE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Z2, Beckman Coulter.
Preparation of pre cultures: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium.
Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

hardness of the final nutrient medium: 1.3 °dH (22.5 mg/L CaCO3)

Test temperature:

21 - 24 °C

pH:

7.8 -8.0

Nominal and measured concentrations:

100 mg/L (nominal)

Details on test conditions:

EXPOSURE CONDITIONS
Test vessels: 300 mL Erlenmeyer flasks with cotton stoppers, test volume: 100 mL
Culturing apparatus: Shaking incubator in which a temperature in the range 21 °C to 24 °C was maintained at +/- 2 °C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily.
Light intensity: A light intensity ranging from 60 to 120 µE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.
Cell density measurements: Cell densities were measured in a microcell counter (Z2, Beckman Coulter) by taking aliquots from each test flask, which were not replaced.
Experimental design: 1 test concentration plus 1 control; 6 replicates per concentration, 6 replicates per control; Initial cell density in the test cultures approximately 5000 cells per millilitre; Additionally highest test concentration without algae.
Test item concentration: 100 mg/L
Duration of exposure: 72 hours
Criteria of effects: The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.022 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The results are expressed in terms of geometric mean measured values.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.022 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The results are expressed in terms of geometric mean measured values.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.022 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The results are expressed in terms of geometric mean measured values.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.022 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The results are expressed in terms of geometric mean measured values.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.022 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: The results are expressed in terms of geometric mean measured values.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.022 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: The results are expressed in terms of geometric mean measured values.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.022 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: The results are expressed in terms of geometric mean measured values.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.022 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: The results are expressed in terms of geometric mean measured values.

Details on results:

No toxic effects against algae were observed at the limit of water solubility under exposure conditions.
Effective concentrations ranged from 0.090 % to 0.13 % of nominal values at 0 hours, ranged from 0.014 % to 0.028 % of nominal values at 24 hours, from 0.0063 % to 0.014 % of nominal values at 48 hours and from 0.0050 % to 0.031 % of nominal values at 72 hours.

Reported statistics and error estimates:

All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10). For the calculations all algae counts were divided by a factor of 10000.

Effective Concentrations (ECx) for Growth Rate at 72 h

Growth Rate of Desmodesmus subspicatus

%Inhibition caused by the test item after 72 h.

Treatm. [mg/L]	Mean	Std. Dev.	n	%Inhibition
Control	1.709	0.0240	6	0.0
100	1.697	0.0316	6	0.7

Overview over the ECs of the Test Item on Growth Rate at 72 h

Effects on Growth Rate

Growth rate (G) and its inhibition relative to control (%I) as computed from the raw data for test intervals selected.

Treatment	0 - 24 h		0 - 48 h		0 - 72 h
[mg/L]	G	%I	G	%I	G	%I
Control	1.885	0.0	1.558	0.0	1.709	0.0
100	1.810	4.0	1.478	5.1	1.697	0.7

Effective Concentrations (ECx) for Yield at 72 h

Yield of Desmodesmus subspicatus

%Inhibition caused by the test item after 72 h.

Treatm. [mg/L]	Mean	Std. Dev.	n	%Decrease
Control	84.019	5.8028	6	0.0
0.02215	80.985	7.7595	6	3.6

Overview over the ECs of the Test Item on Yield

Effects on Yield

Yield (Y) and its inhibition relative to control (%I) as computed from the raw data for test intervals selected.

Treatment	0 - 24 h		0 - 48 h		0 - 72 h
[mg/L]	Y	%I	Y	%I	Y	%I
Control	2.800	0.0	10.802	0.0	84.019	0.0
0.02215	2.560	8.6	9.123	15.5	80.985	3.6

Validity criteria fulfilled:

yes

Conclusions:

The adverse effects of phosphorothioyltris(oxybenzene-4,1-diylcarbamoyloxyethane-2,1-diyl) trisprop-2-enoate on the growth rate show a value of ErC 50 > 0.022 mg/L after 72 hours. No toxic effects against algae were observed at the limit of water solubility under exposure conditions.

Executive summary:

A study was performed to assess the adverse effects of phosphorothioyltris(oxybenzene-4,1-diylcarbamoyloxyethane-2,1-diyl) trisprop-2-enoate on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of phosphorothioyltris(oxybenzene-4,1-diylcarbamoyloxyethane-2,1-diyl) trisprop-2-enoate dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and a folded filter.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to STUDENT-t test for Homogeneous Variances. The following values were determined:

ErC 50 (0-72 h):       > 0.022 mg/L

ErC 10 (0-72 h):       > 0.022 mg/L

NOEC [r] (tα 0.05):  ≥ 0.022 mg/L

LOEC [r] (tα 0.05):   > 0.022 mg/L

No toxic effects against algae were observed at the limit of water solubility under exposure conditions.

The results are expressed in terms of geometric mean measured values. Effective concentrations ranged from to 0.090 % to 0.13 % of nominal values at 0 hours, ranged from 0.014 % to 0.028 % of nominal values at 24 hours, from 0.0063 % to 0.014 % of nominal values at 48 hours and from 0.0050 % to 0.031 % of nominal values at 72 hours.

Description of key information

A study was performed to assess the adverse effects of phosphorothioyltris(oxybenzene-4,1-diylcarbamoyloxyethane-2,1-diyl) trisprop-2-enoate on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a limit test concentration of nominally 100 mg/L of phosphorothioyltris(oxybenzene-4,1-diylcarbamoyloxyethane-2,1-diyl) trisprop-2-enoate dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and a folded filter.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to STUDENT-t test for Homogeneous Variances. The following values were determined:

ErC 50 (0-72 h):       > 0.022 mg/L

ErC 10 (0-72 h):       > 0.022 mg/L

NOEC [r] (tα 0.05):  ≥ 0.022 mg/L

LOEC [r] (tα 0.05):   > 0.022 mg/L

No toxic effects against algae were observed at the limit of water solubility under exposure conditions.

The results are expressed in terms of geometric mean measured values. Effective concentrations ranged from 0.090 % to 0.13 % of nominal values at 0 hours, ranged from 0.014 % to 0.028 % of nominal values at 24 hours, from 0.0063 % to 0.014 % of nominal values at 48 hours and from 0.0050 % to 0.031 % of nominal values at 72 hours.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.022 mg/L

EC10 or NOEC for freshwater algae:

0.022 mg/L

Additional information

ErC50 and ErC10 value should read '> 0.022 mg/L'