2-ethoxy-N-hydroxybenzenecarboximidamide

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Purity not specified
- Batch number: BXR5U4G

Analytical monitoring:

yes

Remarks:

HPLC analysis and UV/VIS-detection

Details on sampling:

- Stock solution: at 0 hours
- Control: at 72 hours
- Test concentrations: 12.5 and 100 mg/L each tested at 0 and 72 hours

Vehicle:

no

Details on test solutions:

Pre-treatment of test item and preparation of test item concentrations:
- 200.2 mg of the test item were added to 2 litre 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 7.9.
- Appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.446 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- 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 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). The growth medium (according to BRINGMANN & KÜHN (1977)) was renewed once a week. Cell density measurements were made using a microcell counter, Sysmex F300, Digitana.
- 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:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

1.3 °dH (22.5 mg/L CaCO3)

Test temperature:

21-24 °C

pH:

7.9 - 8.9

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

- HPLC-values after 72 hours of exposure (mg/L): 12.5 (nominal) equals12.680 (measured); 100 (nominal) equals 100.651 (measured)
- Effective concentrations: from 103 to 113 % of nominal values at 0 hours, and from 101 to 106 % of nominal values at 72 hours

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 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 (Sysmex F300, Digitana) by taking small aliquots from each test flask, which were not replaced.
- Experimental design: 4 test concentrations plus 1 control; additionally highest test concentration without algae
- Replicates: 3 per concentration, 3 per control
- Initial cell density in the test cultures: approx. 5000 cells per millilitre
- Method of administration: stock solution
- Criteria of effects: item-induced inhibition of yield [y], growth rate [r] of the algal population

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

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:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 25 - < 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The results are expressed in terms of nominal concentrations. Effective concentrations ranged from 103% to 113 % of nominal values at 0 hours, and from 101 % to 106 % of nominal values at 72 hours.

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.

Validity criteria fulfilled:

yes

Remarks:

cell density in the control cultures increased by a factor of at least 16 within 72 hours; mean coefficient of variation in the control cultures did not exceed 35 %; coefficient of variation of average specific growth rates did not exceed 7 %

Conclusions:

After 72 hours of exposure NOECr was determined as 50 mg/L, ErC50 as > 100 mg/L for algae Desmodesmus subspicatus. The results are expressed in terms of nominal concentrations. Effective concentrations ranged from 103 % to 113 % of nominal values at 0 hours, and from 101 % to 106 % of nominal values at 72 hours.

Executive summary:

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 (Desmodesmus subspicatus) were exposed for a period of 72 hours to a range of concentrations, nominally 12.5, 25, 50 and 100 mg/L of Imidoxim 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 Williams Multiple Sequential t-test Procedure.

After 72 hours of exposure NOECr was determined as 50 mg/L, ErC10 and ErC50 were both determined as higher than 100 mg/L. The results are expressed in terms of nominal concentrations. Effective concentrations ranged from 103 % to 113 % of nominal values at 0 hours, and from 101 % to 106 % of nominal values at 72 hours. This toxicity study is classified as acceptable and satisfies the guideline requirements for the Algae study.

Description of key information

The study was conducted according to OECD Guideline 201. After 72 hours of exposure NOECr was determined as 50 mg/L and ErC50 as higher than 100 mg/L for algae Desmodesmus subspicatus. The results are expressed in terms of nominal concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

50 mg/L

Additional information

"Should read: 72h-ErC50: >100 mg/L and 72h-NOEC: 50 mg/L"

Effective concentrations ranged from 103 % to 113 % of nominal values at 0 hours, and from 101 % to 106 % of nominal values at 72 hours.