Addition product of dehydrated castor oil, linseed oil and maleic anhydride

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:

From September 19, 2017 to September 29, 2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Preparation
The water-accommodated fractions (WAF) of the concentrations to be tested were prepared. This was done by weighing the nominal loads, adding the appropriate amount of algal medium (demineralised water enriched with minerals but without algae) and shaking for 7 days. After membrane filtration through 0.45 μm PTFE filters, the solutions were used for preparation of the treatments.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Strain: SAG Strain Number 86.81

Origin and Culture: The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Pre-Culture: Four days before the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.7 – 23.4 °C

pH:

6.9 - 8.4

Nominal and measured concentrations:

Nominal concentration in mg/L: 3.2; 10; 32; 100; 320; 1000
Real load in mg/L: 3.2; 10; 32; 100; 324; 1005.2

Details on test conditions:

Performance of the Study:

For each treatment, 200 mL of the respective test substance solution was mixed with the necessary amount of algal pre-culture (0.362 mL) to achieve a cell concentration of 2.7+E3 cells/mL. For the blank control, 350 mL nutrient medium was used instead of test substance solution and mixed with the necessary amount of algal pre-culture (0.634 mL).
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates. In these mixtures, the pH-value was measured. Samples for the analytical determination were taken.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was measured again. At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test substance). The content of DOC in the test vessels was measured at the start and at the end of the test.

Number of replicates: 6 replicates for the blank control and 3 replicates for each treatment

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

790.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

13.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

349.18 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.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

285.35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

13.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

The 72 h EC50s of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1. The details of calculation are stated in chapter 19, Annex 5: Statistical calculation using ToxRat® Professional 3.2.1.

Geometric mean of measured concentrations:

 

Nominal concentration test substance	Geometric mean
mg/L	mg/L
Blank control	-
3.2	0.2
10	1.0
32	2.7
100	13.9
320	77.1
1000	518.2

 

Validity criteria:

 

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	73	Valid
Mean coefficient of variation of daily growth rates	max. 35%	11%	Valid
Coefficient of variation of average growth rate during the whole test period	max. 7%	2%	Valid

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h EC50 of the test substance in fresh water algae were determined to be and 65.50 mg/L (biomass growth) and 349.18 mg/L (growth rate). The 72 h NOEC were determined to be 13.90 mg/L (biomass growth) and 13.90 mg/L (growth rate). The results are expressed as geometric means of measured concentrations.

Executive summary:

A study was conducted to determine the short term toxicity of the test substance to Desmodesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. One valid experiment was performed. The study was conducted under static conditions with an initial cell density of 2.7+E3 cells/ml. Six loading levels of the Water Accommodated Fraction (WAF) were tested ranging from 3.2 to 1000 mg/L (nominal). Incubation time in the test system was 72 h. The cell concentration of each replicate was determined by measuring the cell numbers every 24 h with an electronic particle counter. Growth rate and the yield were determined from the cell number at the respective observation times. At the start and at the end of the test, the concentration of the test substance in the test solutions was estimated by determination of the Dissolved Organic Carbon (DOC) content in the test solutions using a carbon analyser. Significant inhibition of algal growth was observed at the following concentrations: 320 and 1000 mg/L. The correlation between nominal and measured concentration was weak because of the limited solubility of the test substance. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. The 72 h EC50s of potassium dichromate were determined in a separate reference test. Under the study conditions, the 72 h EC50 of the test substance in fresh water algae were determined to be 65.50 mg/L (biomass growth) and 349.18 mg/L (growth rate). The 72 h NOEC were determined to be 13.90 mg/L (biomass growth) and 13.90 mg/L (growth rate) (Muckle, 2017).

Key value for chemical safety assessment

EC50 for freshwater algae:

349.18 mg/L

EC10 or NOEC for freshwater algae:

13.9 mg/L

Additional information

A study was conducted to determine the short term toxicity of the test substance to Desmodesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. One valid experiment was performed. The study was conducted under static conditions with an initial cell density of 2.7+E3 cells/mL. 6 loading levels of the water accommodated fraction (WAF) were tested ranging from 3.2 to 1000 mg/L (nominal). Incubation time in the test system was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate and the yield were determined from the cell number at the respective observation times. At the start and at the end of the test, the concentration of the test substance in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser. Significant inhibition of algal growth was observed at the following concentrations: 320 and 1000 mg/L. Significant inhibition of algal growth was observed at the following concentrations: 320 and 1000 mg/L. The correlation between nominal and measured concentration was weak, because of the limited solubility of the test substance. Therefore, the determination of the results was based on the geometric mean of the measured concentrations. The 72h-EC50s of potassium dichromate were determined in a separate reference test. Under the study conditions, the 72 h EC50s of the test substance in fresh water algae were determined to be and 65.50 mg/L (biomass growth) and 349.18 mg/L (growth rate). The 72 h NOECs were determined to be 13.90 mg/L (biomass growth) and 13.90 mg/L (growth rate). The results are expressed as geometric means of measured concentrations (Muckle, 2017).