Anthranilamide

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Anthranilamide
Physical state/Appearance: Beige powder
Purity: 99.65%
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Storage
The samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 1.0, 3.2, 10 and 32 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (8.0 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP). Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 – 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

1.0mg/L 8.7
3.2mg/L 8.8
10mg/L 8.8
32mg/L 8.7
100mg/L 8.5

The pH value of the control cultures was observed to increase from pH 8.4 at 0 hours to pH 8.7 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Nominal and measured concentrations:

1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.14 x 105 cells per mL. Inoculation of 500 mL of test medium with 8.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS MultitronVersion 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Test Organism Observations
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

Analysis of non-inoculated test preparations at 72 hours showed measured concentrations to be between 80% and 94% of nominal values and hence the decline in measured test concentrations in the inoculated test vessels was considered to be due to adsorption to algal cells. Overall the decline in measured concentrations followed a concentration dependent pattern with greater losses being observed at the lower test concentrations. This effect was considered to be due to there being greater numbers of algal cells in the lower concentrations and hence greater surface area for adsorption to occur.
Whilst there was a significant decline in measured test concentrations over the test period, the test item was shown to be stable in aqueous medium and hence the decline was considered to be due to adsorption to algal cells. As such it may be considered that the algal cells were exposed to nominal concentrations of test item throughout the test period and all results have been calculated in terms of nominal test concentrations alone.

Inhibition of growth rate
ErC10 (0 - 72 h) : 6.0 mg/L
ErC20 (0 - 72 h) : 13 mg/L
ErC50 (0 - 72 h) : 44 mg/L*

Inhibition of Yield
EyC10 (0 - 72 h) : 4.3 mg/L
EyC20 (0 - 72 h) : 5.7 mg/L
EyC50 (0 - 72 h) : 9.3 mg/L; 95% confidence limits 7.9 - 11 mg/L

Observations on Test Item Solubility
At 0 hours all control and test cultures were clear colorless solutions. At the end of the test the control, 1.0 and 3.2 mg/L test cultures were green dispersions, the 10 mg/L test cultures were pale green/yellow dispersions, the 32 mg/L test cultures were very pale green/yellow dispersions and the 100 mg/L test cultures were very pale orange colored solutions.

* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Range-finding Test

The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 91% to 94% of nominal values. A decline in measured test concentration was observed at 72 hours to between 46% and 88% of nominal values. The decline in measured concentrations followed a concentration dependent pattern with greater losses being observed at the lower test concentrations. It was therefore considered that the decline was due to adsorption to algal cells and not instability of the test item.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the nominal test concentrations gave the following results:

Growth Rate
EC50 (mg/L) 44
95% Confidence Limits (mg/L) Not Determined
No Observed Effect Concentration (NOEC) (mg/L) 3.2
Lowest Observed Effect Concentration (LOEC) (mg/L) 10

Yield
EC50 (mg/L) 9.3
95% Confidence Limits (mg/L) 7.9 – 11
No Observed Effect Concentration (NOEC) (mg/L) 3.2
Lowest Observed Effect Concentration (LOEC) (mg/L) 10

Description of key information

In an OECD 201 study, under GLP conditions, the 72 hour toxicity to algae EC50 (growth) of Anthranilamide is 44 mg/L. The NOEC is 3.2 mg/L (Envigo 2016).

Key value for chemical safety assessment

EC50 for freshwater algae:

44 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L