Dimethyl (dimethoxyphosphinyl)succinate

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:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

GC

Details on sampling:

sampling at: 0h, 24 h, 48 h, 72 h
samples after 24 and 48 h were taken but not analyzed, since the results obtained at test start and test end were sufficient for the evaluation of the study

Vehicle:

no

Details on test solutions:

Preparation of the test media
The highest test concentration was prepared by weighing in 100.005 mg of test item and transferring it into a glass bottle. Afterwards, 1 L of growth medium was added and the treatment was stirred for about 30 minutes at room temperature. The other test concentrations were prepared by serial dilution of the highest test concentration with growth medium using a spacing factor of 2.0.

Test performance
For the growth test, four replicates of test cultures were prepared for each treatment level and eight replicates for the control. Every flask was filled with 100 mL of the respective test medium.
The algal pre-culture (461 µL at a cell density of 2.168 x 106 cells/mL) was added to the test vessels to achieve the initial cell concentration of 10,000 cells/mL.
At the beginning of the test, the initial cell concentration was calculated based on the cell number of the pre-culture. During the test, the cell concentrations were determined after 24, 48 and 72 hours.

Test organisms (species):

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

Details on test organisms:

- Origin: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG.
-Cultivation: The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980)). Prior to testing a pre-culture will be established in standard OECD growth medium to obtain exponentially growing algae for the test. The culture duration of the pre-cultures was 3 days.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.0 - 22.5 °C

pH:

The pH of the controls was increased during the test from 7.46 at test start to a mean value (n = 8) of 7.68 at test end. In the test cultures the initial pH in the treatments ranged from 7.39 to 7.51 and were between 7.70 and 7.81 at the end of the test.

Nominal and measured concentrations:

nominal: 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L (spacing factor 2.0).
At test initiation, the measured concentrations were 5.93, 11.71, 23.52, 47.80 and 95.08 mg test item/L (93.7 – 95.6 % of nominal).
After 72 hours, the measured test item concentrations were 5.35, 10.58, 21.05, 42.61 and 85.35 mg test item/L (84.2 – 85.6 % of nominal; 89.1 – 90.4 % of initial).

Since the concentration of the test item satisfactorily maintained within ± 20 % of the nominal and the measured initial concentration throughout the test, analysis of the results was based on nominal concentrations.

Details on test conditions:

Test procedure

Growth medium
The sterilised synthetic growth medium (OECD medium) according to OECD 201 was used as growth medium.
All stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“. The pH of the medium was obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air.
pH, at test start about 8.0

Test vessels
Test vessels were 250 mL conical glass flasks covered with air-permeable silicone-sponge caps. The vessels and caps were sterilized prior to use (autoclaving).

Test conditions
The culture vessels were incubated at a temperature in the range of 21 to 24 °C, controlled at ± 2°C, with a light intensity (OSRAM Standard “cool white”) adjusted to 60 - 120 µE m-2 s-1 (4440 - 8880 lux) close to the surface of the liquid.

The cultures were oscillated by continuously stirring on a laboratory shaker with 150 rpm (Incu-bation Shaker Multitron®, INFORS Typ HT, Switzerland). The light intensity was measured daily using a cosine (2 π) receptor (LI-250A, LI-COR) in µE m-2 s-1 at level of the test media surface.

The solution pH was measured in one test vessel per test concentration including the control. At the end of the test, the solution pH values were measured directly in test vessels. The test culture temperature was measured in one additional test vessel.

- Test concentrations: control, 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L
- 8 replicates/control, 4 replicates/concentration

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: see 'Details on results'

Details on results:

There were no concentration-dependent effects on the growth of the green alga up to the highest test item concentration of 100 mg test item/L after testing the effect with five different test concentrations over an exposure period of 72 hours.

The NOEC for growth rate was determined to be lower than 6.25 mg test item/L. However, due to the low inhibition of 4.24 to 9.13 % at a concentration range of 6.25 to 100 mg test item/L and a missing dose-responserelationship, the NOEC was set to be above or equal to 100 mg test item/L.

Since the concentration of the test item satisfactorily maintained within ± 20 % of the nominal and the measured initial concentration throughout the test, analysis of the results was based on nominal concentrations.

Results with reference substance (positive control):

The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in a non-GLP test twice a year. The latest (December 2017) ErC50 value of 2.90 mg/L (nominal; 95% confidence limits: 2.66 – 3.16 mg/L) for inhibition of growth rate is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.

Reported statistics and error estimates:

Treatment of results
- Since the concentration of the test item satisfactorily maintained within ± 20 % of the nominal and the measured initial concentration throughout the test, analysis of the results was based on nominal concentrations.
- The mean values of the cell counts for each concentration plot of the exposure test were used for plotting growth curves.
- For the growth test, mean average growth rates were calculated (entire exposure period of 0 - 3 d).
- For the growth test, calculation of the percent inhibition compared to controls of growth rate or sectional growth rate [r], and yield [y] for the exposure period were performed according to the guideline
- For the growth test, the percent inhibition values of growth rate and yield were plotted as a function of the test item concentration.
- Since the test results did not show inhibition above 50 % they were not statistically analysed to determine an EC50 and EC10 value together with 95 % confidence intervals.
- The NOEC values were determined using appropriate statistical methods (Multiple Sequentially-rejective Median (2x2-Table) Test After Bonferroni-Holm and Welsh t-test after Bonferroni-Holm Procedure.
- The computer program ToxRat® was used for statistical evaluations.

Microscopic observations were performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae (as may be caused by the exposure to the test substance) at the end of the test.

Microscopic observations of the algae cells at test initiation and test termination.

Date	Nominal test concentration [mg/L]	Observations
Test start, day 0		Normal appearance of inoculum culture for all test concentrations.
Test end, day 3	Control	Normal appearance of algal cells, almost no cell debris.
	6.25	Normal appearance of algal cells, almost no cell debris.
	12.5	Normal appearance of algal cells, almost no cell debris.
	25.0	Normal appearance of algal cells, almost no cell debris.
	50.0	Normal appearance of algal cells, almost no cell debris.
	100.0	Normal appearance of algal cells, almost no cell debris.

Validity criteria fulfilled:

yes

Remarks:

cell number in control increased by a factor >16 in 72 h, mean of replicate coefficients of variations (CV) in the section-by-section growth rate of controls ≤ 35 %, CV of average specific growth rate at test end in replicate control cultures ≤ 7 %

Conclusions:

A study was performed according to OECD 201 to determine the toxicity of the test item dimethyl (dimethoxyphosphinyl)succinate on the growth of the uni-cellular freshwater green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum), which showed no concentration-dependent inhibiting effect on the growth over the range of the tested concentrations (6.25 - 100 mg/L). The respective 72-hour ErC50 was above the highest test concentration of 100 mg test item/L. A NOEC for growth rate of above or equal to 100 mg test item/L was determined.

Executive summary:

A study was performed to determine the toxicity of the test item dimethyl (dimethoxyphosphinyl)succinate on the growth of the uni-cellular freshwater green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum). Exponentially-growing cultures of the green alga were exposed to five concentrations of the substance plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.

Control and the following nominal test concentrations were prepared: 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L reflecting a spacing factor of 2.0. For the examination of the inhibition of alga growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested.

Since the concentration of the test item satisfactorily maintained within ± 20 % of the nominal and the measured initial concentration throughout the test, analysis of the results was based on nominal concentrations.

No concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.

The respective 72-hour ErC50 was above the highest test concentration of 100 mg test item/L. A NOEC for growth rate of above or equal to 100 mg test item/L was determined.

The NOEC for growth rate was determined to be lower than 6.25 mg test item/L. However, due to the low inhibition of from 4.24 to 9.13 % at a concentration range of 6.25 to 100 mg test item/L and a missing dose-response-relationship, the NOEC was set to be above or equal to 100 mg test item/L. Effects below 10 % compared to the control are generally not considered to be ecotoxicologically relevant and it is generally recommended by OECD and EFSA to use the EC10 approach in preference to the NOEC approach for the environmental risk assessment.

Description of key information

A study was performed for 72 h according to OECD guideline 201 to determine the toxicity of the test item dimethyl (dimethoxyphosphinyl)succinate on the growth of the uni-cellular freshwater green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum).

Control and the following nominal test concentrations were prepared: 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L reflecting a spacing factor of 2.0. For the examination of the inhibition of alga growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested.

Since the concentration of the test item satisfactorily maintained within ± 20 % of the nominal and the measured initial concentration throughout the test, analysis of the results was based on nominal concentrations.

No concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.

The respective 72-hour ErC50 was above the highest test concentration of 100 mg test item/L. A NOEC for growth rate of above or equal to 100 mg test item/L was determined.

The NOEC for growth rate was determined to be lower than 6.25 mg test item/L. However, due to the low inhibition of from 4.24 to 9.13 % at a concentration range of 6.25 to 100 mg test item/L and a missing dose-response-relationship, the NOEC was set to be above or equal to 100 mg test item/L. Effects below 10 % compared to the control are generally not considered to be ecotoxicologically relevant and it is generally recommended by OECD and EFSA to use the EC10 approach in preference to the NOEC approach for the environmental risk assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Should read: EC50 >100 mg/L, NOEC >= 100 mg/L