Reaction product of 6H-dibenz[c,e][1,2]oxaphosphorin 6-oxide, itaconic acid and ethylene glycol

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:

2012-05-09 - 2013-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The GLP study was conducted according to an internationally accepted guideline. All study parameters are given in detail.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The pre-culture was adjusted to a cell concentration of about 11*104 /mL instead of 4.5*104 /mL as stated in the study plan.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The temperature during the test was in the range of 24 °C - 26 °C instead of 21 °C -24 °C as stated in the study plan.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

he stock solution prepared in acetone had a concentration of 13808 mg/ instead of exactly 13800 mg/L as stated in the study plan.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The pre culture was prepared from a permanent culture instead directly from the stock culture as stated in the study plan.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

During validation of the analytical method, the maximal solubility in algal test medium was determined as 13.8 mg/L. Acetone was used as solvent to prepare the test solutions of the concentrations needed directly before the start of the test.

Stock solutions containing the nominal treatments 13800, 6300, 3000, 1400 and 600 mg/L in acetone were prepared. The highest stock solution (13808 mg/L) was prepared by direct weighting of test item, whereas the lower treatments by dilution if the highest one.

The concentration of the stock solutions was 1000-fold higher than the respective concentration in the treatments (13.8, 6.3, 3.0, 1.4 and 0.6 mg/L resp. 100, 46, 22, 10 and 4.6 % of the estimated maximal solubility in the algal test medium). For the solvent control, acetone in a concentration of 1 mL/L was used.

Note: concentration of the solvent acetone surpassed the recommended solvent concentration (as stated in OECD document no. 23: "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"). It was proven in several experiments, though, that a concentration of 1 mL/L is tolerated by the algae.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Specification: Unicellular freshwater green alga.

Genus, Species: Desmodesmus subspicatus

Strain: Chodat

Family: Scenedesmacea

Order: Sphaeropleales

Origin and Culture:

The culture of Desmodesmus subspicatus was obtained in Oct. 2011 by MBM Science-bridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared..

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 - 26 °C

Nominal and measured concentrations:

for details see table below

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 11 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.:

> 11 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: AUC

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: AUC

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: AUC

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

The EC50s of potassium dichromate were determined in a current reference test. For the estimation of the EC50s of the positive control, the fits showed sufficient statistical corre-spondence of the data with the dose-response-equation.

Biological Results of the Positive Control Parameter Value 95%-confidence-interval NOEC72h 0.25mg/L not determinable LOEC72h 0.4mg/L not determinable 72h ErC50 1.40mg/L > 1.1 mg/L 72h EbC50 0.61mg/L 0.51 - 0.74 mg/L 72h EVC50 0.38mg/L 0.30 - 0.47 mg/L

Growth Rate p, Area under the Curve AUC, Yield Main Study

Nom. Conc.	Measured Conc. 0 h	%NomConc.	Measured Conc. 72 h	%NomConc.	%Recov­ery	geom. Mean.
mg/L	mg/L	%	mg/L	%	%	mg/L
Control	< LOQ	-	0.10	-	-	-
Solvent Control	< LOQ	-	0.07	-	-	-
0.6	0.62	103.23	0.23	37.84	36.66	0.38
1.4	1.55	110.69	0.83	59.39	53.65	1.14
3.0	3.18	106.12	1.73	57.74	54.41	2.35
6.3	6.72	106.64	2.36	37.49	35.16	3.98
13.8	15.09	109.36	7.72	55.97	51.18	10.80

LOQ=Limit of quantification(0.069mg/L)

Validity criteria fulfilled:

yes

Conclusions:

The following results for the test item Ukanol FR 70 were determined:
Endpoint NOEC LOEC EC50
Growth Rate > 11 mg/L > 11 mg/L > 11 mg/L
AUC > 11 mg/L > 11 mg/L > 11 mg/L
Yield > 11 mg/L > 11 mg/L > 11 mg/L

Executive summary:

The Toxicity of Ukanol FR 70 towards Desmodesmus subspicatus was determined following OECD guideline 201.

One valid main study was performed. The study was performed using five concentrations ranging from 0.6 to 13.8 mg/L, resp. from 4.6 to 100 % of the maximal water solubility in algal test medium. The maximal solubility in test medium was determined during validation of the analytical method. As the test item is poorly soluble in water, the concentrations tested were prepared using acetone. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the solutions at 440 nm every 24 hours with a spectral photometer. The cell density of the cultures was calculated based on the correlation curve between the adsorption and the cell density of the cultures determined by microscope counts. Growth rate area under the growth curve (AUC1) and the yield2 were determined from the cell densities at the respective observation times. No significant inhibition of algal growth was observed in the control, the solvent control and in any treatment. During validation of the analytical method, the test item had been stable in algal medium over a period of 72 hours (recovery 103 %). Nevertheless, analytical determinations showed that the test item was not stable under test conditions in this study. The measured concentrations lay between 103 % and 111 % of the nominal concentration at the beginning of the test and between 38 % and 59 % of the nominal concentration at the end of the test. The recovery after 72 hours was in a range of 35 % and 54 % of the measured start concentration. The unexpected poor recovery means that the characteristic of the test medium in presence of the algae is not identical with the medium without algae, used during validation of the analytical method. Without much doubt, the loss of test item was caused by ingestion by the algae. Therefore, determination of the biological results was based on the geometric mean of the measured concentrations. The EC50s of potassium dichromate were tested in a current reference test. The values lay within the normal range of the laboratory.

Description of key information

The following results for the test item Ukanol FR 70 were determined:

Endpoint       NOEC       LOEC       EC50

Growth Rate       > 11 mg/L       > 11 mg/L       > 11 mg/L

AUC       > 11 mg/L       > 11 mg/L       > 11 mg/L

Yield       > 11 mg/L       > 11 mg/L       > 11 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

11 mg/L